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Characterization of wall teichoic acids in two morphological forms of Arthrobacter crystallopoietes

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Title:
Characterization of wall teichoic acids in two morphological forms of Arthrobacter crystallopoietes
Creator:
Hellmuth, John Hardin, 1952-
Language:
English
Physical Description:
viii, 81 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Amino acids ( jstor )
Amino sugars ( jstor )
Cell walls ( jstor )
Chromatography ( jstor )
Enzymes ( jstor )
Morphogenesis ( jstor )
Phosphates ( jstor )
Polymers ( jstor )
Sugars ( jstor )
Teichoic acids ( jstor )
Arthrobacter crystallopoietes ( lcsh ) ( lcsh )
Bacterial cell walls ( lcsh ) ( lcsh )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Microbiology and Cell Science thesis Ph. D
Teichoic acids ( lcsh ) ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 74-80.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by John Hardin Hellmuth.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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05313034 ( OCLC )
AAK0600 ( NOTIS )

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CHARACTERIZATION OF WALL TEICHOIC ACIDS
IN TWO MIORPH~OLOGICAL FORM'S OF
Arthr~obac-Cer crystal Lopoietes











By

JOHNi HARDINU HELZLMUTH


A DISSERTATIONu PRESENTED TO THEF GRADUATE COUNCIL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUTIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY





UNIVERSITY OF FLOR.IDA















ACICTOWLEDGS~~EMNT`S


The author wishes to thank sincerely Dr. Edward Previc for his

encouragement, suggestions, and criticisms during the preparation of

this dissertation.

He also wishes to thank the other members of his supervisory

committee, Dr. Arnold Bleiweis, Dr. David Hubbell and Dr. Lonnie

Ingram for the help they contributed in the preparation of this

manuscript.

The author would like to express his gratitude to the other

members of the Department of Microbiology and Cell Science for

generously supplying equipment and materials during his work.

Special thanks to Steven Hurst for technical assistance with

amino acid analyses.

Appreciation is extended by the author to his parents for the

encouragement they gave him to pursue his education.

Finally, the author is particul.arly indebted to his wife, Mlartha,

for patience, encouragement, and assistance during his graduate work.





TABLE OF CONTENTS


Page

.ii


ACKNOWJLEDGEMENTS.. ...

LIST OF TABLES .........


. . . . .


LIST OF FIGURES .... .. .. . .. . . . vi

ABSTRACT . .. .. .. . ..... .. .. . vi

INTRODUCTION . .. .. .. .. .. .... .. . 1
Taxonomy .. ... ... .. .. .. .. . . 1
Control of Mlorphogenesis . ... .. .... .. .3
Justification for This Study ... .. .. .. .. .. 5
Teichoic Acid Background .. .... .. .. .. .. 6


MATERIALS AND METHODS .........
Organism . . . . . . .
Media . . . .
Growth . . . .
Cell Wall Isolation ......
Purification of Cell Wall. .
Teichoic Acid Isolation ...
Analysis of Purified Walls ...
Paper Chromlatography..
Analytical Procedures .....
knino Acid and Amino Sugar Analysis
Teichoic Acid Purification ..
Gel Filtration .. .......
Chain Length Determination


. . I . .


Determination of Miolar Ratios of Glycerol
Phosphate in Purified Teichoic Acids
Characterization of Alkaline Hydrolysate


. . 19
. . 19


RESULTS . . . . . . . . . . . . . .
Growth of Arthrob~a-ter cryst~allopoietes ATCC 15481 ....
Almino Acid and kmino Sugar Analysis of
Purified Wall Hydrolysates ..............
TCA Extraction of Purified Walls .,,..,,......
Recoveries of Purified iNall and TCA Extractable
Material (Crude Teichoic Acid) ............
Characterization of Acid-Hydrolyzed TCA Extracts
by Paper Chromatography ...............


22
22

22
25

29

29









Page

Amino Acid and Amino Sugar Analysis of
Crude Teichoic Acid .. .. .. .. .. . . . 35
Crude Teichoic Acid Purification on DEA1E-Sephadex . .. 35
Purified Teichoic Acid Characterization on
Sephadex G-100 .. .... .. .. . .. . . 39
Chain Lenght Determination .. .. .. . . . . 39
Molar Ratios of Glycerol and Phosphate in
Purified Teichoic Acids . ... .. . . . 43
Characterization of Alkaline Hydralysates .. .. . 43

DISCUSSION . . .. .. ... . . . . . . 52
Peptidoglycan Composition .. .. .. .. . . . 52
TCA Extraction .... .. .. .. . . . . 57
Characterization of Acid Hydrolysates .. . ... . 59
k-mino Acid and Amino Sugar Analyses of Teichoic Acid .. 61
DEAE Chromatography .. ... . .. . . . . . 61
Acid Hydralyses of Purified Teichoic Acids . .. .. . 62
Gel Filtration .. .. .... .. . ... . . 63
Determination of Chain Lenght .. .. .. . . . 63
Glycerol-Phasphate Ratios ,,. . .. .. . . . 65
Alkaline Hydrolysis .. .... . .. .. . . . 66
Summary of Teichoic Acid Structure . .. .. . . . 68
Possible Roles for Teichoic Acids in Arthrobacter .. 68

LITERATURE CITED .... .. . ... . . 74

BIOGRAPHICAL SKETCH .. .. ... .. .. .. . . 81















LIST OF TABLES


Table Page

1 Carbohydrate constituents of representative
teichoic acids . .. .. .. . . .. 8

2 Molar ratios of amino acids and amino sugars in
walls digested with various enzymes .. .. .. .. 26

3 Paper chromatography of acid-hydrolyzed TCA1
extracts. I. Phosphoric acid esters .. .. .. .. 31

4Paper chromatography of acid-hydrolyzed TCA
extracts. II. o-Glycols .. .. .. .. .. 32

5 Paper chromatography of acid-hydrolyzed TCA
extracts. III. Sugars (external standard) . ... 33

6 Paper chromatography of acid-hydrolyzed TCA
extracts. IV. Sugars (internal standard) .. .. .. 34

7 Malar ratios of labile and total phosphate in
alkaline phosphatase-digested, purified
teichoic acids ... ... .... . .. .. . 42

8 Molar ratios of glycerol to inorganic phosphate . .. 44
















LIST OF FIGURES




Figure Page

1 Growth of Arthroba~ccter cryistallopoietes .. .. 24

2 TCA extraction of purified walls . .. .. ... 28

3 DEAE~-Sephadex chromatography of crude teichoic
acid .. .. .. .. .. .. ... .. .. 38

4 Sephadex G-100 chiromatography of purified
teichoic acid .. .. .. .. .. .. .. ... 41

5 Alkali-hydrolyzed teichoic acid elution on D)EAE-
Sephadex . .. .. .. . .. . 47

6 Sephadex G-50 chromatography of neutral and cationic
components of alkali-hydrolyzed GSTA .. .. .. 49

7 C~i-Sephadex chromatography of neutral and cationic
components of alkali-hydrolyzed GSTA ... .. 51















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



CHARACTERIZATION OF WALL TEICHOIC ACIDS
IN TW~O MORPHOLOGICAL FORMS OF
Arthrobactear c~rystairopoiete s


By

John Hardin Hellmuth

December 1978

Chairman: Dr. Edward P. Previc
Major Department: Mlicrobiology and Cell Science

The cell wall teichoic acid isolated from two morphological forms

of Ar-t~r~obacter c~rystal3opoletes is characterized. Cell walls purified

from spherical (GS) cells contained 18.2 llg of phosphorus per ag of cell

wall, while those from rod-shaped (LS) cells contained 21.2 pg phosphorus

per mg of cell wall. Trichloroacetic acid extracts of purified walls of

both forms were found to contain poly (glycerol phosphate) with hexosamine

glycosidically attached. In GS teichoic acid there was 2SZ as much

hiexosamine as glYcerol phosphate and in LS teichoic acid there was

21% as much. The hexosamine included at least 50% N-acetylated glucos-

amine and galactosamine in about a 5-to-1 ratio. Evidence is presented

which suggests that the hexosamine may- exist as trisaccharide side

chains. Chain lengths were estimated by the racio of tacal phosphate

to alkaline phosphatase-labile phosphate. By chis menchd, ceichoic

acids from GS-grown cells had an average length of 38 glycerol phosphate


vii








units and those from LS-grown cells had an average length of 70

units. The possible significance or these findings as they relate

to mrorp~hogenesis in Arthrobacter is discussed.





INTRODUCTION


Taxonomy



The genus Arthrrobactetr is characterized by cells which can undergo

nutritionally-controlled sphere-rod morphogenesis (48). The species

.rthrobacter crys~callopoietes was first described by Ensign and

Rittenberg (30i) who isolated the organism by enrichment cultures con-

taining 2-pyridone. The brilliant green crystalline pigment produced

by this species growing on solid medium containing 2-pyridone was

later identified by Kuhn et al. (57) to be a hydrate of the mono-

potassium salt of 4,5,4',5'-tetrahydroxy-3,3'-diazadiphenoqin-

(2,2'). Recently, Kolenbrander and Weinberger (52) found that A.

crystailopo~ietes lost the ability to produce pigment from 2-pyridone

at a high spontaneous frequency of 0.26% loss per generation. This

high spontaneous loss has also been observed in the strain of A.:

oryssablopoidese used in this dissertation research. Kolenbrander and

Weinberger (52) present good evidence that the loss of a plasmid is

correlated with loss of a'oility to produce pigment, In the present

study this presented a problem since the plasmid-less strain seemed

to grow faster than the parent strain in glucose-salts media. It was

therefore necessary to minimize che number of accumulated mutants by

starting each culture for harvest from a single pigment producing

colony.





According to Bergey's Manual (48), the name Arthrobacter crysbai-

Lopoietes is a probable subjective synonym of Arthrobacter globiforrmis.

The Manual also states that the salient feature distinguishing A.

corystatZZopoietes from A. globiformis is the ability to utilize

2-pyridone as a sole carbon and energy source and to produce a crystal-

line pigment from it. It thus seems likely that A. crystallopoiet~es

which has lost the plasmid has almost exactly the same phenotype as

A. globifobreds. Since the presence or absence of the plasmid may

affect the wall composition by either direct effects (e.g., plasmid

genes might modify wall synthesis) or indirect effects (e.g., plasmid

presence might affect growth rate which might in turn affect wall

synthesis), comparisons between the walls of these bacteria can not
be made on the assumption that A. --rysIallopoietes and A. globiformi

are the same bacteria.

The nutritional control of morphology in Al. crystallopoietes was

first demonstrated by Ensign and Wolfe (31). They showed that expo-

nential growth of spheres could be obtained in a defined. medium con-

taining glucose. Exponential growth of rods could be obtained by

adding certain morphogenesis-inducing compounds to the defined glucose

medium. This idea forms the basis for the methods of obtaining spheres

and rods in the following work. The only difference is that the rod-

inducing medium contains lactate but no glucose. Since there is a

diauxic suppression of glucose catabolism and anabolism in the presence

of rod-inducing compounds (54), this difference is minimized.









Controlof M~orphogenesis



Although the morphology of Arthrobacter can be extrinsically con-

trolled by nutritional means, this control is only indirectly related

to the unknown intrinsic control by the bacteria. The intrinsic

control is more directly related to growth rate which can be controlled

independently from the type of nutrition. Luscombe and Gray (60) have

done this by growing a strain of Arthrobacter under carbon-limiting

conditions in a chemostat. They found that, at 250C, rods were only

produced at dilution rates above 0.25 per hour. At rates lower than

this, the cells were always spherical. This raises a problem because

it means that whenever rod and sphere-shaped arthrobacters are compared,

morphology is not the only difference; growth rate also changes and

hence could affect many variables. This is a fundamental problem

which makes it difficult to establish correlations as causal rela-

tionships and it applies to this study. Hamilton et al. (39) have

sidestepped this problem by isolating a spherical morphological

mutant of A. O~mystailopoieees which is unable to undergo sphere-rod

morphogenesis but increases its growth rate in rod-inducing media.

The genetic nature of this lesion has not yet been reported.

The intrinsic control of morphogenesis probably consists of a

chain of events, some of which are genetic, some enzymatic, and some

structural. Certain changes at any level of this chain can probably

manifest themselves at the morph~ological level. Therefore, any one

event should not be said to control morphogenesis. Nevertheless,

several authors (39,47,53,61,68,69,73) claim that their effect which





correlates with morphology is probably the factor which controls

morphogenesis in A. crystallopoietes.

It has been shown that Ai. crystallopoietes contains two RNA poly-

merases both of which have considerable and almost equal synthetic

capabilities in in vitro studies (47). This correlates with another

finding by the same lab (61) that morphogenesis involves differential

transcription of the DNA with some transcripts being present all the

time and others being present only at certain times during the morpho-

genetic cycle. Furthermore, St. John and Ensign (73) by using RNA

and DNA synthesis inhibitors were able to show that morphogenesis can

occur in the absence of DNA replication but RNAT synthesis is required

for morphogenesis to occur.

Hamilton et al. (39) suggest that the level of cyclic adenosine

3',5'-monophalsphate (c-AEIP) may be important in regulating morpho-

genesis. The levels of e-ANT were shown to rise steeply at times just

prior to the initiation of shape changes (either sphere-to-rod or rod-

to-sphere). This suggested that elevated c-APhe levels may act as the

"trigger" which induces the cells to change shape. Their morphological

mutant (mentioned above) did not show these changes.

Krulwich et al. (53, 55) have shown that the glycans in the pepti-

doglycan of spheres are generally shorter than those of rods. This

change was also correlated with a change in the activity of a wall-

bound N-acetylmuramidase. They implied that morphology, therefore,

depends on glycan chain length.

Previc (68) has suggested that morphology in most bacteria may

be determined by the presence or absence of extra crossbridges invol-

ving free carboxly groups of diaminopimelic acid (Dpm) and other









tetrafunctional amino acid groupings (e.g., lysylasparty1 crossbridges

in some Lactobacilius species). In a mutant of A. erystallopoietes

Previc and Lowell (69) have shown that spherical mutants contain lysine

in the penultimate position of tetrapeptides while rod-shaped mutants

contain Dpm. Transitional stages during sphere-to-rod morphogenesis

show a gradually increasing amount of Dpm present. Thus, morphology

in this strain may be dictated by the presence or absence of the extra

carboxyl group of Dpm.

Several authors seem to agree that a rod shaped morphology requires

a more rigid peptidoglycan than a spherical morphology (37,55,68).

Ward and Claus (84) have found that for A. crystallopoietes the rod

peptidoglycan layer is thinner than the sphere peptidoglycan. If the

rod peptidoglycan is more rigid than the sphere peptidoglycan, then

this difference must be due to changes in the peptidoglycan structure

rather than simply a thickening of the wall.

It should now be apparent that there are changes during morpho-

genesis at each step in the chain of events which regulates morphology.

What is still not apparent is how all these changes fit together to

produce morphogenesis. Of course, some of the changes may not be

related to morphagenesis at all but only reflect altered growth rates.

So, the question of how morphogenesis occurs is still unanswered.



Justification for This Study



The original intent of this author was to determine how peptido-

glycan might help to maintain the different shapes of A. crystailopoietes.

To accomplish this the composition of the wall was examined for changes





which might correlate with morphology. During the beginning of the

quantitative analyses of the components of the wall, there arose

certain evidence that a wall teichoic acid might be present. The

determination of its qualitative and quantitative structure has become

the primary topic of this dissertation. Although this seems far re-

moved from the question of morphogenesis, there m~ay be an intimate

association between the two. These possibilities are examined in the

Discussion.



Teichoic Acid Background



Teichoic acids are molecules which occur in nearly all gram-positive

bacteria (3). These molecules are grouped into two categories de-

pending on their cellular location and their structure; lipoteichoic

acids are found associated with the cell membrane and wall teichoic acids

are associated with the peptidoglycan (4).

Lipoteichoic acids are found in most gram-positive bacteria. These

molecules are all of the same structural type, i.e., they consist of

a linear backbone of poly (glycerol phosphate) which is linked by

phosphodiester bonds involving C-1 and C-3 of adjacent glycerol

phosphates (3). Diversity of lipoteichoic acid structure is introduced

by various carbohydrate side groups (see Table 1) which are attached

at the C-2 hydroxyl group. D-Alanine is usually found as an ester

linked either to the C-2 hydroxyl of glycerol phosphate or to glycosyl

hydroxyl groups. It is though that all lipoteichoic acids are

covalently attached to glycolipid in the cell membrane (51).










Wall teichoic acids are more structurally diverse than lipo-

teichoic acids. The classical wall teichoic acids are polymers of

glycerol or ribitol phosphate. Both types can have various carbo-

hydrate side groups (see Table 1) and/or D-alanine. These polymers

are thought to be cavalently attached to the peptidoglycan in the cell

wall. Other acidic polymers have been found in the walls of gram-

positive bacteria which resemble the classical teichoic acids. Polymers

of glycerol phosphate and one or more sugars have been found in a

number of cases and polymers of sugar phosphates are also known.

Both types of polymers are similar to teichoic acids in that they

convey a net negative charge to the outer surface of the cell and,

therefore, may have functions similar to the teichoic acids (51).





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MATERIALS AND METHODS


Organism



.4rthzrobacter crystallopoietes ATiCC 15481 was obtained from the

American Type Culture Collection. In order to maintain the presence

of the plasmid which bears the gene(s) responsible for green crystal

production from 2-pyridone, stock cultures were frequently streaked

on 2-pyridone-containing plates (30) and pigmented colonies were

picked for further subculturing. All inocula for cultures to be

harvested were also checked in this manner.



Media



Defined media contained, per liter: 1.73 g K2HP04, 2.33 g KH2PO4,

1.00 g (NK4)2SO4, 5.0 g D-glucose (GS) or sodium lactate (LS),

0.5 g MIgSO4, and 10.0 ml of a trace salts solution. The trace salts

solution contained, per liter: 1.5 g nitriloacetic acid, 0.5 g

MnS04, 1.0 g NaCl; FeCl2, CaC12, CoCl2, and ZnSOq, 0.1 g each, and

CuS04, A1K(SO4)2, H3BO3, and (NH4)6Mo7024, 0.01 g each. The trace

salts solution was adjusted to pH 7.0 with 1.76 g NaOH. The glucose,

MIgSO, and the trace salts solutions were each autoclaved separately (31).

A complex pigmentation medium contain 0.2% 2-pyridone, 0.05%

yeast extract, and inorganic salts was prepared according to Ensign and

Rittenberg (30).









Growth



All cultures were agitated at 300C in a New Brunswick Gyrotory

shaker-incubator. Optical density (0.D.) was followed at 450 nm

on a Beckman DLC-2 spectrophotometer with a 1.0 cm light path. Optical

densities were maintained between 0.05 and 1.0 0.D. units by periodic

transfers to fresh media.



Call Wall Isolation



Exponential cultures were passed through coiled copper tubing which

was submersed in an ice-water bath. The cooled effluent was directed

into a De Laval Gyro Tester Laboratory centrifuge with the bowl

prechilled to 40C. The time for culture liquid to pass through the

cooling coils and out the centrifuge spout was measured using enough

nigrosin dye added to a deionized water run to serve as a visual

marker. The temperature and 0.D. at 450 am of' the final effluent

were also measured during bacterial harvests. From these measurements

it was concluded that cultures were cooled from 300C to 8-100C in

approximately one minute and that 95% or more of the culture's bacterial

mass was removed from the effluent as determined by 0.D. measurement.

All subsequent handling of bacterial samples was at no higher than

40C unless otherwise specified. Whole cell pellets were washed twice

by resuspension in deionized water and centrifugation at 13,000 x g

for 30 min in a Servall GSA rotor. Bacteria were suspended in

deionized water at a concentration no greater than 50 mg dry wt/ml and

broken in a Braun homogenizer at 4,000 rpm for 4 minutes using 0.11 mm





diameter glass beads. Tributyl phosphate (0.5% v/v) was used to reduce

foaming. Glass beads were removed first by filtration on fritted glass

filters and then by centrifugation at 160 x g for 30 min. Crude cell

walls were then pelleted from the supernatant at 27,000 x g for one hour.



Purification of Cell Wall



The method of Braun and Sieglin (14) for wall purification was

modified by an additional sodium dodecyl sulfate (SDS) extraction

before treatment with pronase (69). Crude wall pellets were suspended

in 0.1 M~ ethylenediaminetetraacetic acid (EDTA), pH 7.4 (at least

20 ml/g of whole cell dry wt), and then pelleted at 27,000 x g, washed

and resuspended in deionized water. Suspensions were added dropwise

to stirred, boiling 4% SDS (at least 15 ml/g of whole cell dry wt).

These mixtures were allowed to cool to room temperature with stirring

overnight. These mixitures were then centrifuged at 27,000 x g for

one hour. Pellets were resuspended in deionized water and reextracted

with boiling 4% SDS. These pellets were washed with deionized

water three times and resuspended in 0.05 M Tris, pH 7.4 (at least

5 ml/g of whole cell dry wt). Pronase was added to a final concen-

tration of 100 ug/ml and the mixtures were digested at 370C for 16

hours. The walls were centrifuged for one hour at 37,000 x g. The

resultant pellets were reextracted by boiling SDS as above and

rewashed three times with deionized water. The final pellets were

lyophilized for dry weight determinations.





Teichoic Acid Isolation



To release teichoic acid (t), purified walls were extracted with

10% (w/v) tricl0roacetic acid (TCA) at 4oC for 24 hours. Walls were

pelleted from this mixture at 27,000 x g for one hour and the pellets

were reextracted with fresh 10% TCA at 4oC for 24 hours. Again fresh

cold TCA was exchanged for supernatant TCA and the extraction was

continued for 38 hours. Crude teichoic acids were collected either

by precipitation from the TCA with absolute ethanol (5 volumes) at

-200C and subsequent centrifugation at 13,000 x g for 30 minutes, or

by removing the TCAl by ether extraction to pH 4 and subsequent lyo-

philization.



Analysis of Purified Wlalls



Purified walls (0.4 mg/ml) were hydrolyzed in 4 N HC1 at 1050C for

11 hours. The excess acid was then removed by evaporation, followed

by three cycles of deionized water additions and reevaporations. The

residues were dissolved in 0.01 N HCI (1 mg wall/ml acid) and then

filtered through 0.45 um Millipore filters. Unsolubilized material

was present in negligible amounts. The filtrates were analysed for

amino acids and amino sugars on a JEOLCO Automated Amino Acid

Analyzer (76) .





Pagpe Chromatography



Whatman papers no. 1, no. 4, and no. 3 MM were soaked for at least

30 minutes in a solution which was 0.1 NT in acetic acid and 0.1 M

in EDTA. This solution was than washed out by suspending the papers

in deionized water and then removing excess water by decantation. A4t

least ten sequential water washes of increasing duration were performed,

with a final wash of at least one hour. Washed papers were allowed

to dry in horizontal stacks at 300C. Papers to be eluted parallel

to the grain of the paper were cut to 23 cm x 57 cm with the grain

running parallel to the long axis of the paper. Papers to be eluted

perpendicular to the grain were cut to 23 cm x 46 cm with the grain

running perpendicular to the long axis. Samples were spotted 7 cm

from the top of the papers by repeated application of approximately

0.25 pl each time, then drying by hot air. The papers were mountain-

folded along a line at 6 cm and valley-folded along a line at 3 cm

from the top of the papers. Thnis folding allowed the papers to be hung

from a glass trough in a pyrex tank (30.5 em x 30.5 cm x 61 cm). The

papers were allowed to equilibrate with the vapor phase of the eluting

solvent in a glass chromatography tank for at least 2 hours before

the eluting solvent was added for the beginning of elution. All

chromatograms were eluted at ambient temperature, typically ranging

from 220-27oC. The solvent systems for elution used were: (Al) n-propanol-

ammonia (28-30%)-water (6:3: 1) (4t0) and (B) ethyl acetat e-pyridine-water

(10:;:3) (36).









Analytical Procedures



Phosphate Aissays


Total phosphate was assayed by the ascorbic acid-molybdate method

developed by Chen et al. (23). The ashing method of Lowry et al. (58)

was used for convenience in handling large numbers of samples.

Inorganic phosphate was measured by the Chen method as modified

by Aimes (1). KH2P04 (anhydrous analytical reagent, Mallinckrodt)

was used as a standard in both total and inorganic phosphate methods.

Chromic acid-washed tubes were used for all phosphate assays.


Amino Sugar Assays


N-Acetylamino sugar s were measur ed by the borate-p-dime thyl-amino-

benzaldehyde (DMAB) method of Reissig et al. (70).

N-Acetylglucosamine (Sigma) was used as the standard for this assay.

Amino sugars were assayed for N-acetylation with acetic anhydride by

a method similar to the N-acetylamino sugar assay as described by

Ghuysen et al. (35). This method primarily detects amino sugars

which are free in the C-1 position but it also detects amino sugars

which are covalently bound at that position. The extinction coefficient

for the latter reaction, however, is more than thirty fold smaller than

that for the former reaction. D-Glucosamine-HC1 (K+K Laboratories, Inc.)

was used as a standard for this assay.

Total amino sugars could be detected if an acid hydrolysis (2 N HC1,

1000C, 3 hours) followed by neutralization preceded the N-acetylation

step of the amino sugar assay. The same standard was used as for the

amino sugar assay.





The minimum amount of N-acetylation of amino sugar was estimated

by a method suggested by Ellwood et al. (29). This method involves

a mild acid hydrolysis (0.1 N H2S04, 100oC, 30 min in a sealed

ampoule) which breaks the glycosidic linkage while leaving most of

the N-acetyl groups intact. After hydrolysis, a slight excess (10%)

of the amount of BaCO3 necessary for neutralization was added. The

resulting BaS04-BaCO3 mixture was removed by low speed ce-ntrifugation.

N-Acetylhexosamines and total amino sugars were then determined for

the neutralized samples.


Carbohydrate Aggayg


Simple sugars, oligosaccharides, and polysaccharides with either

free or potentially free reducing groups were determined by the phenol-

H2SO4 method of Dubois et al. (28). D-Glucose (analytical reagent,

Mallinckrodt) was used as a standard.


Protein A Eg[


Protein was measured by the Lowry method (59) which involves

first a reaction of protein with Cu+2- under alkaline conditions and

then reduction of a phosphomolybdate-phasphotungstate reagent by the

copper-treated protein. Bovine serum albumin (Sigma) was used as

a standard.





Enzymatic Determination of Glycerol


Free glycerol was determined by using the Glycerol Stat-Pack

(Calbiochnem). This assay employs the following reaction sequence:

(1) glycerol + adenosine triphosphate (ATP) glycerol kinase

a-glycerophosphate + adenosine diphnosphate (ADP)

(2) ADP + phosphoenolpyruvate pyruvate kinase pyruvate + ATP

(3) pyruvate +- nicotinamide adenine dinucleotide (reduced) (NADH)

lactate dehydrogenase lactate + nicotinamide adenine

dinucleotide (oxidized)

The d isappearanc e of NADH was followed s pec tropho tome tr ically at a

wavelength of 340 nm using the same instrument used for 0.D. readings.

Glyceral (analytical reagent, Mallinckrodt) was used as a standard.



Amino Acid and A~mino Sugar Analysis



Crude teichoic acid samples (GS, 5.0 mg; LS, 5.6 mg) were each

hydrolysed in 1.0 ml of 2N HC1 at 1000C for 3 hours. The hydrolysates

were vacuum dried over Na0H pellets and then redissolved in 1.0 ml

of deionized water. Portions of these (200 pl each) were diluted in

0.01 N HC1 to 2.2 ml (final concentrations: GS, 0.45 mg/ml; LS,

0.51 mg/ml). These samples were subjected to amino acid and amino

sugar analysis in the same way as described for purified wall hydro-

lysates.





Teichoic Acid Purification


Crude teichoic acids were purified on approximately 9 g of DEA1E-

Sephadex A-50. One cm of Sephadex G-25 course gel was used at the

bottom of the column as bed protection. The DEAE-Sephadex was swollen

and loaded in 0.1 M NaC1. The bed diameter was 2.5 cm and the bed

length varied depending on ionic strength (35.5 cm at 0.1 M NaCl and

24.0 cm at 1.0 M NlaC1). The bed was then washed with 500 ml of

0.1 M1 NaC1.

Teichoic acids were eluted by an increasing gradient of NaC1.

For the LS preparation, 200 ml of a 0.1 M 0.5 M gradient was

followed by a 200 ml gradient from 0.5 M to 1.0 M2 NaC1. A total of

160 fractions of approximately 2.1 ml each was collected. For the GS

preparation, 500 ml of a 0.1 M 1.0 M Nacl gradient was used of elu-

tion. A total of 95 fractions of approximately 5.3 ml each was collect-

ed. The fraction numbers for the GS elution profile ini the Results

section have been normalized with the LS profile using equivalent salt

concentrations.



Gel Filtration


Gel filtration was carried out on Sephadex G-100. Purified teichoic

acids (GS, 42 mg; LS, 6 mg) were applied to a column bed of dimentions:

2.5 em x 31 cm. Total bed volume was 160 al. The void volume was

determined to be 54 ml by using Blue Dextran 2000 (Pharmacia, Average

molecular weight, 2 x 106). A Blue Dextran sample (1 ml of a 0.2%

solution) was applied to a column and the effluent was collected in





approximately 3 ml fractions. These fractions were monitored for

0.D. at 260 nm as a measure of Blue Dextran. The number of the

fraction with the highest 0.D. was multiplied by the fraction volume

to obtain the void volume. Eluant was deionized water. A constant

pressure head of 31 cm was maintained during loading and running.

A Gilson automatic fraction collector was adjusted to collect 95

drop (approximately 3 ml) fractions.

Results were graphed in terms of Ilmoles of total phosphate vs.

K. K is defined as: Ve V,
K ----
V, Vo

where: V, = void volume

Ve = total bed volume

Ve = elution volume



Chain Length Determination


In two separate determinations, about 1 mg and 0.5 mg of each

type (GS and LS) of purified teichoic acid was diluted from concen-

trated solutions to 125 ill with deionized water. Then, 125 ul of

0.04 M (NH432CO3 and 10 p~l (0.6 mg or 7.2 units) of Escherichia coli

alkaline phosphatase (60 mg/ml, 12 units/mg, Worthington Biochemical

Corporation, Code: BAPSF bacterial alkaline phosphatase salt

fraction) were added. One unit is defined as that activity liberating

one umale of p-nitrophenal per minute at pH 8.0 and 250C. The mixture

was incubated in a slowly shaking 370C water bath for 18 hr. The

samples were then analyzed for inorganic and total phosphate content.

The ratio of total to inorganic phosphate content was taken as an

approximation of chain length.





Determination of Malar Ratios of Glycerol and Phosohate

in Purified Teichoic A~cids



Purified teichoic acid samples were hydrolyzed in 2 N HC1 at 1000C

for 4 hours. Acid was removed by vacuum evaporation or lyophilization.

The samples were then digested with alkaline phosphatase by the same

general method described under chain length determination. The

digested samples were then analyzed for free glycerol, inorganic and

total phosphorus. Because of phosphorus present in the enzyme

preparation, it was also necessary to run enzyme blanks with no

added sample and to subtract out the blank values from the sample

values. This method was tested on a known concentration of 3-glycero-

monophosphate which produced a molar ratio of glycerol to phosphate

of 0.997.



Characterization of Alkaline Hydrolysate



A purified teichoic acid sample (GS, 32.6 llmales total phosphate)

was hydrolyzed in 1.45 ml of 1? M a0H at 1000C for 3 hours. The hydro-

lysate was neutralized with 300 pl of 4 N HCL. The final pH was

close to pH 7.0 as judged by pH paper. The neutralized sample was

diluted with deionized water to 14.5 ml to attain a salt concentration

of 0.1 M NaC1.

The diluted sample was loaded an the same DEAE-Sephadex( A-50

column used for the teichoic acid purufication. In preparation for

this run, the column had been previously washed with 500 ml of 1 M

NaC1. The washed column was slowly reswollen with a 1.0 M 0.1 M





NlaC1 gradient of 500 ml. Finally, the column was washed with another

500 ml of 0.1 M NaCl for final equilibration. Once loaded, the sample

was washed with 500 ml of 0.1 M NaC1. The wash affluent was collected

and lyophilized. The adherent portion of the sample was eluted

from the column with 500 ml of a 0.1 M 1.0 MI NaC1 gradient.

Fractions (100, approximately 4 ml each) were collected. These

fractions were assayed for total phosphate and total amino sugars.

The lyophilized wash sample was then filtered on a Sephadeex

3-50 column (diameter 1.6 cm, height 62 cm, 124 ml bed volume, 56 ml

void volume) with deionized water in order to resolve low-molecular

weight, non-anionic fragments. Fractions (65 of 3 ml each) were

collected and analyzed for total and inorganic phosphate and total

amino sugars. All phosphate-con gaining fr-ctions were then pooled

and lyophilized. This material (approximately 2.8 g) was assumed

to be mostly NaC1 and was diluted with deionized water to 0.1 MI

NaC1 on that basis.

This diluted sample was applied to a CM-Sephadex C-50 column

(approximately 1.9 g of gel; column with following dimensions:

diameter, 1.5 em, length, 30 cm) to further resolve cationic molecules.

The column bed was then washed with 50 ml of 0.1 M NaC1. After

washing, the remaining sample was eluted with 250 ml of a 0.1 M -

1.0 M NaCl gradient, and 46 fractions (5.4 ml each) were collected.

These fractions were analyzed for total phosphorus and total amina

sugars. The two peaks (two fractions/peak) containing significant

amounts of hexosamine were each combined and lyophilized. These

lyophilized samples were triturated in the presence of absolute





ethanol (10) The ethanol fractions were then evaporated to dryness

and the residues were triturated again with absolute ethanol (1 ml) .

The ethanol-soluble portions were again evaporated to dryness. The

residues were each dissolved in 450 Fpl of deionized water and then

analyzed for free glycerol.





RESULTS


Growth of Arthrobacter crustatlovoietes

ATCC 15481



Typical growth curves are shown in Figure 1. Generation times

were: GS, 10.8 hr + 0.5 br; LS, 3.5 hr + 0.2 hr.


Amino Acid and Amino Sugar Analysis of Purified Wall Hydrolysates


It was observed that cell walls purified according to the protocol

in Materials and Methods usually contained certain non-peptidoglycan

amino acids. The following experiment was an attempt to remove these

amino acids using proteolytic enzymes.

Cell walls were isolated from lyophilized cells (GS or LS, 300

mg each) and were purified as described in Material and Methods

with one modification. Just before the pronase digestion, each

sample was divided into four equivalent portions. One portion of

each was not treated with any enzyme and served as the control;

the second portions were treated with pronase (500 ug/ml); the

third portions were treated with trypsin (100 ug/ml); and the fourth

portions were treated with a-chylmotrypsin (100 ug/ml). All digestions

were carried out in 0.05 M Tris, pH 7.4 at 37"C for 16 hours. Following

the enzymcatic digestions, each sample was treated as described in

Materials and Miethods for the rest of the wall purification and





Figure 1. Growth of Arthrobacter crysa~tcZcpoletes. GS (7), LS (V).





E 4
O

.3-




.2-








5 10 li
time, hours








amino acid analysis. Peak areas on chromatograms were computed by

a JOELCO integrater. Relative concentrations were then computed

using known areas of standard amino acids and sugars. These relative

concentrations were then divided by the relative concentrations for

the glutamic acid peak on each chromatogram. The values for these

ratios are given in Table 2. These analyses indicate that the normal

peptidoglycan amino acids are present in the expected ratios. They

also show that trypsin is the most effective enzyme in removing

the trace amino acids,

Chemical analysis of purified cell wall hydrolysates also shows

the presence of significant amounts of phosphorus (GS, 18.2 pg ?/mg

wall; LS, 21.2 ug P/mg wall). Since Krulwich and Ensign (55) had

reported phosphorus in their cell wall preparations of A. orystaZlo-

poisfes and had found that all of it could be removed 'ay hot TCA

extraction, a similar experiment was carried out with the cell walls

prepared in this laboratory.



TCA Extraction of Purified Walls



Purified cell walls (GS, 19 mg; LS, 18 mg) were suspended in

10 ml of cold 10% TCA and were stirred with a magnetic stirring bar

at 40C. At various times, 1.0 ml portions were removed and extracted

three times with petroleum ether (B.P. 300C) to remove the TCA. The

sample was then centrifuged at 27,000 x g for a half hour.

Total phosphate was determined for both the soluble and insoluble

fractions as shown in Figure 2. Phosphate was extracted by TCA with















Molar ratios of amino acids and amino sugars in walls digested
with various enzymes.a




Treatment Control Pronase Trgy -hmtysin
GS LS GS LS GS LS GS LS

GlcKH2I, 1.42 1.53 1.65 1.48 1.59 1.58 1.58 1.44

Lys 0.88 0.94 1.00 0.92 0.94 0.95 0.96 0.86
Asp 0.06 0.04 0.04 0.03 ND 0.01 03.03 0.03
Thr 0.03 0.03 0.03 0.02 ND T 0.04 0.02
Mur 0.45 0.47 0.45 0.44 0.44 0.46 0.48 0.48
Glu 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1,00

Gly 0.04 0.04 0.03 0.02 ND 0.01 0.03 0.02
Ala 2.61 2.64 2.61 2.68 2.75 2.68 2.68 2.68
Val 0.03 0.03 0.02 0.01 ND ND 0.02 0.02

Glcr`HW, 1.29 1.28 1.32 1.30 1.33 1.40 1.33 1.41
GalNHY+ ++d


TAlBLE 2


(a) Mlolar ratios are relative to glutamic acid.


vN not detectable. T


trace, + present but amount not determiined.

(b) Glucosamine calculated from short column data from amino acid analyzer.

(c) Glucosamine calculated from long column data.


(d) Gal~M, galactosamine.

















GN -

CIUO
> Li


E OH
m -f~
mu
me 4
m0 0
ar CO

Ec0:





>OUV



~Or






oa



L30
m O


C CL


weu




3?c3



YOM
a3 L

Y .0
00L
a)O *
p no




















O





O
'u,




O



O
E


O





O


IIDM Bw/B~' 'd








time as indicated by the increase in TCA-soluble phosphate. This

increase was paralleled by an equivalent decrease in wall associated

phosphate as indicated by the TCA-insoluble curves.



Recoveries of Purified Wall and TCAi Extractable Mlaterial

(Crude Teichoic Acid)



Valls purified according to the scheme outlined in Material and

Methods yielded 12-14% wall dry weight to cell dry weight for GS

cells and 13-15% for LS cells. In each individual case the LS

cells yielded slightly more wall (6-10% more) on a percentage basis

than the GS cells.

Recoveries of TCA-extractable material varied depending on

whether ethanol precipitation or ether extraction was used to

remove TCA from the samples. When ethanol precipitation was used,

TCA extractable material recoveries (as crude TA dry weight

percentage of purified wall dry weight) were: GS, 21%; LS, 18%.

When ether extraction was used the recoveries were much higher:

GS, 38% and LS, 28%.



Characterization of Acid-Hydrolyzed TCA Extracts by Paper

Chromatography



To determine the nature of this phosphorus containing compound,

acid hydrolyses were performed. The conditions used have been re-

ported to degrade teichoic acids of either polyglycerophosphate or

polyribitol phosphate and substituted with either amino acids or

carbohydrate (3).




J1U


TCA extracts from two separate preparations were hydrolyzed in

2 N HC1 at 1000C for 3 hours. The residues were dried in vacuc

over Na0H and then redissolved in deionized water. Approximately

25 lug of each sample was spotted on each of two washed Whatman #:4

papers along with standards. The papers were then run with solvent

system A perpendicular to the grain. One paper was run ascending

for 32 hours. The other paper was run descending for 5 hours. Both

papers were air dried. Phosphoric acid esters were detected by the

acid-molybdate spray (40). The results of these chromatograms are

given in Table 3. The unknown spots had mobilities characteristic
of 1,2-diphosphoglycerol.


Approximately 50 ug of hydrolyzed TCA extract were run under

descending conditions in solvent system A but, in this case,

a-glycols were detected with periodate-Schiff spray reagent (9).

The results are given in Table 4. By detecting a-glycols the acid

hydrolysate was shown to contain glycerol and not ribitol.

The acid hydrolysates of teichoic acid were also examined for sugar

content. About 50 ug of each sample was spotted on washed Whatman #1

paper along with appropriate standards. The chromatogram was run

with an ascending front of solvent system B for 18 hours. After

air drying, sugars were detected by the silver nitrate-Na0H spray

(19,82). The results are shown in Table 5. The unknowns in this

case show mobilities characteristic of glucose and probably gluco-

samine.

To confirm the identity of glucose in the unknowns, glucose was

added to each unknown as an internal standard: on similar chromatograms.

The results are shown in Table 6. These results tend to confirm the

presence of glucose in acid-hydrolyzed crude teichoic acids.























Rf
Standards Ascending Descending


1,2 diphosphoglyceral 0.14c 0.28c
(50 nmoles) 0.09c 0.16"

a-glycerophosphate 0.24 0.39
(100 nmoles)

Q-glyceraphosphate 0.31 0.39
(100 nmoles)

hydrolyzed cardiolipind 0.35b
(50 males) 0.12


samples

GSTA (25 Fug) 0.13 0.11
0.09

LSTA (25 ug) 0.14 0.14
0.10


(a) Elution was with solvent system A and spots were detected with the
acid-molybdate spray reagent for phosphoric acid esters.

(b) faint blue spot:

(c) dark blue spot

(d) Hydrolysed cardiolipin was used for a 1,3-diphosphoglycerol standard.

(e) not run


TABLE 3

Paper chromatography of acid-hydrolyzed TCA extracts.a
r. Phosphoric acid enters.





















Standards Rf


a-glycerophosphate 0.42
(600 nmoles)

hydrolyzed ribital 0.65
(65 nmoles)

glycerol 0.75
(1 mole)


Samples

GSTA (50 pg) 0.73b

LSTA (50 ig) 0.74b


(a) Elution was descending with solvent system A and spots were
detected using the periodate-Schiff spray reagent for a-glycols.

(b) Allso had yellow spots at the origin which were the only spots
to develop color slowly.


TABLE 4

Paper chromatography of acid-hydrolyzed TCA~ extracts.a
II. a-Glycols






















Standards Rluob


Glucose 1.00
(5 ug)

Galactose 0.88
(5 lig)

Glucosamine 0.39
(5 yg)

Galactosamine 0.30
(5 ug)


sa pres

GSTA (50 ulg) 1.01
0 -0.53c

LSTA (50 us) 1.10
0 -0.53c


(a) Elution was ascending with solvent system B and spots were detected
by the silver nitrate-Na0H spray reagent for sugars.

(b) Rglcs = cm sample spot migrated/cm D-glucose migrated.

(d) Tailing which included darker areas at R = 0.42 and 0.32.
glucose
These darker areas were also reactive witn a DMAB spray reagent (ll).


TABLE 5

Paper chromatography/ of acid-hyidrolyzed TCA extracts.i
III. Sugars (external standards).






















Standard R
glucose


Glucose 1.00


samples

GSTA +- glucose 0.94d
0 -0.42

LSTA + glucasee 0.98d
0 -0.43


(a) Elution was ascending with solvent system B and spots were detected
by the silver nitrate-Na0H spray reagent for sugars.

(b) Rglcs = cm sample spot migrated/cm D-glucose migrated

(c) To 50 pg of each sample spot, 5 ug of D-glucose was added as an
internal standard.

(d) Tailing which included darker areas similar to those in Table 5.


TABLE 6

Paper chromatography of acid-hydrolyzed TCA extracts.a
IV. Sugars (internal standards)








Almino Acid and Amino Sugar Analysis

of Crude Teichoic Acid



When crude teichoic acid samples were subjected to amino acid

and amina sugar analysis, the only major detectable peaks corresponded

to glucosamine and galactosamine. In both types of teichoic acid

samples (GS and LS), the galactosamine was a minor but not negli-

gible amount of the total hexosamine (GS, 18%; LS, 16%) There

were traces of alanine in both samples, but there was too little

to be quantified by the present method.

These data raised the question of whether the carbohydrate found

in acid hydrolysates was cavalently attached to the presumed

teichoic acid or not. Since the carbohydrate material consisted

of neutral or cationic sugars, it was decided that the teichoic acid

could be purified by DEAE chromatography. By allowing the anionic

teichoic acid to stick to the DEAE groups, the carbohydrate, if

not covalently linked, could be washed through the column. Then the

teichoic acid could be recovered by eluting with a NaC1 gradient.



Crude Teichoic Acid Purification on DEAE-Sephadex



Crude teichnoic acids (GS, 142 mg; L~S, 100 mg) were loaded on

DEAE-Sephadex and washed with 0.1 M NaC1. A gradient of NaC1 was

used for elution (for specific details see Materials and Methods).

The washings and eluate were examined for the presence of phosphorus,

reducing groups, and N-acetylhexosamine. The washings showed

negligible amounts of either acid-molybdate or borate-Db:AB reactive









material. There was, however, some phenol-H2SO4 reactive material

present. This amounted to about 5 iymoles of reducing groups for

both GS and LS. The eluate profiles are shown in Figure 3. No

reducing groups were found in these fractions. The major portion

of the phosphate-hexosamine peaks eluted at NaC1 concentrations of

0.73 M 0.82 M for the GS preparations and 0.85 H 0.91 M for the

LS preparation. Both preparations had a smaller amount of material

which eluted at much lower NaC1 concentrations. Fractions 66-80

for LSTA and 43-60 for GSTA were pooled, dialyzed, and lyrophnilized.

Dry weight recoveries were: GS-45.1 mg (32% of total material

loaded on column) and LS-6.6 mg (7% of total material). To tal

phosphate recoveries were GS-70% (109 umoles recovered from 156 ~males

applied to column) and LS-77% (109 Ilmoles recovered from 140 ymoles

applied).

These purified teichoic acids were analyzed for total amino sugars.

This analysis showed that there were 28% as many amino sugar residues

as phosphate residues for the GS teichoic acid and 21% as many for

the LS teichoic acid. By using a mild acid hydrolysis, it was

possible to release the amino sugars with some of the N-acetyl

groups originally present still intact. This approach yielded

47% (GS) and 54% (LS) of the total amino sugar as N-acety1 hnexosamine.

To further characterize the teichoic acids, they were analyzed

by gel filtration.








































Figure 3. DEAE-Sephadex Chromatography of Crude Teichoic Acid

Crude taichoic acids were applied to 9 g of DEA1E Sephadex.
The column was washed with 500 ml of 0.1 M NaCL. A salt
gradient from 0.1 M to 1.0 M NaC1 was used for elution.
Fractions were assayed for total phosphate and N-acetyl-
hexosamine.





1.0 I 0
GSTA



u8






l) 20 30 40 50 60 70
fraction no.


fraction no.





Purified Teichoic Acid Characterization

o 1 Sephadex G-100



Purified teichoic acids (GS-42 mg, LS-6 mg) were run on Sephadex

G-100 Fine in deionized water. The results of these runs are shown

in Figure 4. K values for peak fractions were: 0.20 for LSTA and

0.71 and 0.69 for two separate isolations of GSTA (0.69 profile

not shown).

These results suggested that the molecular weights of the teichoic

acids might be relatively high and that the LS teichoic acid was

substantially larger than the GS teichoic acid. To test this hypo-

thesis, the average chain length was estimated.



Chain Lengthg Determination



Purified teichoic acid was digested with bacterial alkaline

phosphatase at pH 9.5 for 18 hours at 370C to release terminal phos-

phate groups. The ratios of total phosphorus to labile phosphorus

from two determinations are given in Table 7. Labile phosphorus

was also measured for untreated purified teichoic acid and was

not detectable.

These results confirmed the prediction made by gel filtration

that LS teichoic acid at about 70 glycerol phosphate units is, on

the average, larger than GS teichoic acid at 38 units.

With these basic characterizations accomplished, it was necessary

to confirm the polyglycerol phosphate backbone structure by showing

that a one-to-one ratio of glycerol and phosphate existed.








































Figure 4. Sephadex G-100 Chromatography of Purified Teichoic Acid.

Purified teichoic acids were filtered through 160 ml of
Sephadex G-100. Deionized water was used for elution.
Total phosphate was measured for each fraction and
plotted versus K values computed for the corresponding
fractions.




41





6GSTA

-5
E

S4
E




1


.1 .2 .3 .4 K.5 .6 .7 .8 .9 1.0



6LSTA

5

4

E3

2

1


.1 .2 .3 .4 K.5 .6 .7 .8 .9 1.0
























Sample Experiment number Total: labile phosphate


Purified GSTA 1 38

2 38


Purified LSTA 1 66

2 74



(a) Teichoic acids were digested with bacterial alkaline phosphatase.
After digestion inorganic and total phosphate were determined.

(b) Values were determined by: Mlolar concentration of total phos-
phate/molar concentration of inorganic phosphate in digestion
mixture.


TABLE 7

Miolar ratios of labile and total phosphate in
alkaline phosphatase digested, purified teichoic
acids .a








Molar Ratios of Glvcerol and Phosphate in

Purified Teichoic Acids



Purified teichoic acids were acid hydrolyzed to break down the

glycerol phosphate backbone into glycerol mono- and di-phosphates,

free glycerol, inorganic phosphate, and free amino sugars. The

mixture was then treated with alkaline phosphatase. This mixture was

assayed for free glycerol, inorganic and total phosphate. The

molar ratios of glycerol to inorganic phosphate for two determinations

are shown in Table 8. Analysis of total phosphate in these digested

samples showed that alkaline phosphatase released 99% for GS and

87% for LS of the total bound phosphate.

With this preliminary confirmation of the poly (glycerol phosphate)

nature of the backbone it remained to be proven that the amino sugars

present were, in fact, covalently attached to the polyglycerol

phosphate. This was accomplished by alkaline hydrolysis which

breaks phosphodiester linkages but not glycosidic linkages. The

general procedure used was similar to that of Van de Rijn and Bleiweis

(83).



Characterizations of Alkaline Hydrolysates



Purified teichoic acid (GSTA only) was base hydrolyzed under

conditions such as to break phosphodiester linkages without destroying

glycosidic bonds. After neutralization, this hydrolysate was loaded

onto a DEAE column to separate anionic compounds from cationic

and neutral compounds. After the latter compounds were washed from






















Sample Mlolar ratio (glycerol/Pi)b


Lyophilizedc Dessicatedd

GS 0.86 0.86

LS 0.81 0.85



(a) After acid hydrolysis and acid removal, teichoic acids were digested
w~ith alkaline phosphatase and free glycerol and inorganic phosphate
were then determined.

(b) determined by dividing the molar concentration of glycerol in the
digest mix by the solar concentration of inorganic phosphate.

(c) Determination where acid was removed by repeated (3 times) lyophili-
zation and resuspension in small amounts of deionized water.

(d) determination where acid was removed by vacuum evaportion.


TABLE 8

:iolar ratios of' glycerol t~o inorganic phosphate.a





the column, the anionic compounds were eluted using a linear sale

gradient. The profile of this elution is shown in Figure 5. The

total phosphate represented in this profile was 58% (19 moles)

of the total phaosphate (32.6 moles) that was loaded on the column.

The total amino sugar recovery from the elution was 87% (7.9 Ilmoles)

of the total amino sugar (9.1 Ilmales) that was loaded an the column.

The ratio of total amino sugar to phosphate in the single peak of

the total amino sugar was about 3.2 for ten fractions with the most

amino sugar.

The wash fraction contained the other 42% of the phosphate and

12% of the amino sugar. This fraction was further characterized

by running on a Sephadex G-50 column. This profile is shown in

Figure 6. The phosphate containing fractions (#36 #61) were

pooled and lyophilized. More than 99% of the phosphate and the amino

sugar sa~s recovered from the gel filtration column. The lyophilized

sample was diluted to 0.1N M aC1 and applied to a CM-Sephadex column

in order to separate cationic molecules from neutral ones. After

washing, the sample was eluted with a salt gradient and the profile

is shown in Figure 7. Phosphate was also measured but none was

detected (<10 nmoles/mi). Almino sugar was, however, 100% recovered.

The two amino sugar peaks, the pooled wash, and a fraction midway

between the two amino sugar peaks were analyzed for glycerol. The

pooled wash and the midway fraction contained no detectable glycerol.

The smaller of the amino sugar peaks (fractions #;4 and #5) contained

one glycerol for every 2.9 amino sugar residues while the larger

peak (fractions #41 and #42) contained one glycerol for every 6.5

amino sugar residues.





































Figure 5. Alkali-Hydrolyzed Teichoic Acid Elution on DEAE-Sephadex.

Alkcali-hydrolyzed GSTA was applied to 9 g of DEAE-Sephadex.
Nonadherent compounds were removed with a 500 ml wash of
0.1 3 NaC1. Adherent compounds were eluted with a linear
0.1 Y to 1.0 MI NaCl gradient. Fractions were assayed for
total phosphate and total amino sugars.





























o
~150 E

o



o
'100.5
E


O


fraction no.







































Figure 6. Sephadex G-50 Chromatography of Neutral and Cationic
Components of Alkali-hydrolyzed GSTA.

The wash fraction from DEAE chromatography of alkali-
hydrolyzed GSTA was run on 124 ml of Sephadex G-50.
Deionized water was used for elution. Fractions were
assayed for total (Pt) and inorganic (Pi) phosphate,
and total amino sugars.




49










E
O







E

o


2-





2~0 4~0 6~0
fraction no.





























*He7
6000 ~

r De U
o C



US"
L0 $4L
rdZE
F *





r CL C E



LO *H




UO U



O *~





IT~ 0~


3 O
nic
OZ 0~
is2 O
C- umi
a* ?
mC 3



































O cY) cY

(iu/sa(owuL0I'~D6ns ou!ur~ I~lol


















Peptidoglycan Comoosition



Amino acid and amino sugar analyses of purified wall hydrolysates

first suggested the presence of wall associated polymers other than

peptidoglycan in the wall of Arthrobacter crys'-attopoietes. The

boiling SD)S extraction used to purify walls is a very rigorous method

which should remove all noncovalently associated cellular materials.

Despite this, small amounts of the amino acids aspartic acid, threonine,

glycine, and valine were detected (Table 2). These amino acids are

not usually found in the pentapeptide of peptidoglycan. Also, there

was more glucosamine than could be accounted for by peptidoglycan

structure and there was galactosamine present which has not been re-

ported to occur as part of the peptidoglycan.

K~rutwich et al. (55) have also reported on the amino acid and

amino sugar composition of walls purified from A. crystatt~opoietes 15481.

TIhey also found small amounts of aspartic acid (0.15 mole/mole relative

to glutamic acid in spheres, none found in rods) and glycine (0.14

mole/mole in spheres, 0.04-0.05 mole/mole in rods). They did not find

valine or threonine but did find serine (0.13 mole/mole in spheres, none

in rods). It is conceivable that serine and threonine might be

confused in some amino acid analyzer programs since these amino acids

are similar in structure. On the program used for my experiments,

serine and threonine did elute next to each other but they were


DISCUSSION









distinctly different peaks on standard runs. Krulwich et al. (56)

used C~naZaropsis B enzyme (N,0O-diacetylmuramidase) (41), lysos taphin

(a mixture containing endo-N-acetylglucosaminidase and a peptidase

which releases N-terminal alanine and glycine from cell walls of

Starphylococcus auireus strain Copenhagen, hydrolyzes polyglycine cross-

bridges, and, to a lesser extent, N-acetylmuramyl-L-alanine linkages)

(15), and AL~-1 (a peptidase which breaks peptide crossbridges and

N-acetylmuramyl-L-alanine linkages) (79) in separate experiments to

obtain soluble fragemnts which were separated by ECTEOLA cellulose

and gel filtration. N-terminal and C-terminal amino acids were then

determined for various fragments. By these analyses, they came to

the conclusion that aspartic acid and serine were not connected with

either peptidoglycan or teichoic acid. They suggested that these

amino acids were either contaminants or part of a small additional

peptide in the wall. According to their results, the glycine present

was a part of some of the crossbridges in spherical populations. The

glycine present in rod populations was not, however, part of the

peptidoglycan and presumably could also be a contaminant or part of

an additional peptide. Tipper et al. (79) working in the same lab

as Krulwich et al. also have reported on the cell wall composition of

spherical A. orystallopoieees. The composition they obtained is

very similar to that of Krulwich et al. except that Tipper et al. re-

ported about 0.03 pmole/mg of wall of aspartic acid, serine, threonine,

leucine, histidine, and methionine. Since their walls contained 0.42

moless of total glutamic acid/mg of wall, 0.03 umole/mg of wall would

be 0.07 moles/mole of glutamic acid. The nature of these trace









amino acids and the ones which I have found is unclear but their

presence calls for more research dealing with this problem.

Krulwich et al. (55) found additional glucosamine (spheres, 2.09

mole/mole of glutamic acid; rods, 1.74-2.01 mole/mole) ,galactosamine

(spheres, 0.31 mole/mole; rods, 0.25-0.30 mole/mole),unidentified

phenol-sulfuric acid-reactive material (spheres, 1.95 mole/mole;

rods 1.04-1.37 mole/mole),and phosphate (spheres, 2.02 mole/mole; rods,

1.88-2.12 mole/mole). Tipper et al. (41) also reported similar

results for glucosamine and galactosamine.

The walls analyzed in our laboratory also showed extra glucosamine

(Table 1), although the amount was generally less than that found

by Krulwich et al. (55) or Tipper et al. (70). Quantitative differences

in accessory polymers between the present data and past data are to

be expected since, (1) the media used differ and, (2) the strains

of bacteria used could be different because of extensive subculturing.

Galactosamine was also found by our lab but it was not quantitated in

this experiment since at the time the identity of that peak was

unknown. Phenol-sulfuric acid-reactive material was also found in

TCA extractable material but it was found that this material could

be separated from teichoic acid by chromatography on DEAE-Sephadex.

It seems likely that this material may contain the glucose which

showed up in paper chromatography of acid hydrolysates of TCA extractable

material.

Krulwich et al. (55) were able to extract all the phosphate

present in walls with 100% TCAl at 600C in less than one hour for

both sphere and rod wall preparations. They concluded that this

phosphate mustbe contained in teichoic acid since teichoic acids

are extractable under those conditions. There are, however, other









accessory polymers which have been discovered since the paper of Krulwich

et al. (55) and which are extractable under conditions milder than

those of Krulwich et al. (5,8,51,65). Tipper et al. (79) also found a

phosphate polymer which co-eluted with some of the polysaccharide solu-

bilized by MYyxobacter AL-1 enzyme. Nevertheless, Krulwich et al. have

turned out to be correct in their assumption even though today their

data would not be considered sufficient to establish the presence of a

taichoic acid as opposed to a sugar 1-phosphate polymer or a phosphor-

ylated polysaccharide. Krulwich probably realized this later because in

a paper a year after the one described above, Krulwich and Ensign (53)

refer to the same moiety only as a "phnosphate-containing polymer of the

cell walls".

Krulwich et al. (55) were correct about the lack of amino acid sub-

stitution of the teichoic acid. They found amino acids in their TCA

extract but they were in the same ratios as were found in the peptido-

glycan. They also incubated their purified cell wall in two alkaline

buffers (pH 9.2 and pH 10.5) and found that no amino acids were released.

This indicates that no amino acids are ester-linked to either polyols or

to polysaccharides. In this work amino acid and amino sugar analyses of

crude teichoic acids showed almost no amino acids present at all. The

trace of alanine present could account for no more than one residue for

every 100 glycerol phosphate residues in either GS or LS teichoic acid.

It is more likely that this alanine arose from peptidoglycan contamina-

tion.

Experimental data (see Table 2) suggest that all four non-penta-

peptide amino acids can be removed if the right protease is used.

This confirms the hypothesis that these amino acids are not part of









the pentapeptide. This leaves the possibilities that these amino acids

are contaminants, part of normal crossbridges, or part of a peptide

attached to peptidoglycan.

Trypsin is known to be a specific endopeptidase which breaks peptide

bonds in which the carbonyl group is donated by lysine or arginine.

Most peptidoglycan basic structures are notoriously resistant to

trypsin (74), even those which contain penultimate L-1ysine in the

tetrapeptide (64). The reason for this resistance is not clear but

certainly those L-lysine residues which are involved in crossbridges

would not be susceptible due to a lack of positive charge on the

E-aminO grOup which is a necessary condition for trypsin digestion

(49). MIy data indicate that trypsin has very little if any effect

on the level of alanine in purified cell walls. The alanine ratios

remain fairly constant regardless of which proteolytic enzyme is

used. The alanine ratios reported here agree fairly well with those

of Krulwich et al. (55). The alanine in excess of the two moles in

the tetrapeptide probably is due to the L-alanine crossbridges

proposed by Krulwich et al. (55). By C-termninal analysis, they

estimated that from 60 to 70% of the peptides are crosslinked, which

also agrees with the amount of excess alanine reported here.

If the effectiveness of the different proteolytic enzymes is

compared, trypsin seems to be the most effective in removing the

trace amino acids. The fact that trypsin removed most of these

amzino acids while a-chymotrypsin and pronase did not, may indicate

that cleavage of a specific linkage to peptidoglycan is necessary

to remove a small peptide. Also, it had earlier been noticed that

if the concentration of pronase-digested wall to be analyzed was








increased tenfold, a broad spectrum of non-peptidoglycan amino acids

appeared. This would seem to indicate that the trace amino acid re-

present only the most abundant amino acids present and, therefore,

are the only ones normally detected since the levels of these non-

peptidoglycan amino acids were so 10w. Whether or not trypsin can

remove or lower the amounts of these other amino acids is unknown.

The possibility that the trace amino acids are no more than

contaminants from membrane protein can not be discounted. This

problem warrants further research. It is possible that by using a

N-acetylmuramidse to solubilize the peptidoglycan, these amino acids

could be characterized as belonging either to a high or low molecular

weight fraction on gel filtration.

perhaps the most important fact in this experiment is that extra

glucosamine and galactosamine were present. Combined with the presence

of significant amounts of phosphate, this suggested the possibility

that a teichoic acid was present. Since Krulwich et al. (55) had

found that phosphate could be TCA extracted, a similar kinetic

experiment was carried out but using the milder temperature (4cC as

compared to 60oC that Krulwich et al. (55) used) normally used to

extract teichoic acid with the least degradation (8,51).



TCA Extraction



As was shown in the Results section, the initial rate of extraction

of phosphate was relatively high and was almost the same for GS

and LS. By only 8 hours, about 73% of the total extractable phospahte

had already been solubilized. These initially high rates of extraction




36


then dropped dramatically so that another 67 hours were required to

remove the remaining 27% of the extractable phosphate. Thus, the

average rate of extraction for the first 8 hours was about 9% of the

extractable phosphate per hour whereas the average rate after 8 hours

was about 0.4% per hour. This amounted to more than a 22-fold differ-

ence. The explanation for this difference in rates is not known but

it does seem to indicate that there may be two different types of

reactions occurring. After 75 hours of extraction, 81% of the GS and

53% of the LS total phosphate was solubilized. If the slow rate of

extraction remained constant, it would take 5 days for GS and 8 days

for LS phosphate to be completely extracted. Ghuysen et al. (34)

reported that it took 3 weeks to extract more than 95% of the teichoic

acid of Stapu~hyli ocoocus autreus. In the light of that study, it is

likely that the rate of extraction does not remain constant but

decreases further still with time.

The actual dry weight of teichoic acid recovered after 96 hours of

extraction varied depending on the method used for recovery of

teichoic acid from TCA supernatants. If the weights recovered are

corrected for the percentage recovery, then the actual weight of

teichoic acid in the native wall (GS or LS) may be as high as about

43% (crude TA dry weight percentage of purified wall dry weight).A

small proportion of this crude teichoic acid is contaminating poly-

saccharide, so the actual figure is probably closer to around 40%.

The amounts found in certain other gram positive bacteria range from

20% to 30% of the dry weight of the wall (8).









Characterization of Acid Hydrolysates


To d determine whether or not this pho sphat e-containing polymer was

a teichoic acid, acid hydrolyses were carried out under conditions suf-

ficiently rigorous to degrade a glycerol or ribitol teichoic acid to

its monomers (3).

Table 3 shows the phosphate-containing monomers of acid-hydrolyzed

crude teichoic acid. The ascending technique seems to give the least

ambiguous results. The hydrolyzed teichoic acid spots matched fairly

well with the mobility of 1,2 diphosphoglyceral. The fact that there

were two spots in both the standards and the hydrolysates is probably

due to some inorganic phosphate which should migrate a little farther

than 1,2 diphasphoglycerol (50) and exists as a minor contamination in

the 1,2 diphosphaglycerol standard. Hydrolyzed cardiolipin also showed

a pattern of light and dark spots because this compound upon hydrolysis

should give rise to 1,3 diphosphoglycerol (Rf, 0.12) and glycerol mono-

phosphates (Rf, 0.35). These mobilities agree well with those found in

the literature (7,29,50). The hydrolyzed teichoic acid spots on the

descending run make it more difficult to choose between the 1,2 and the

1,3 compound. Indeed, the hydrolyzed teichoic acid samples may be a

mixture of both which runs together on paper chromatography. This is

actually what one would expect since a certain amount of ester migra-

tion can occur under acidic conditions (3). It is noteworthy that no

glycerol monophosphates were detected but the reason for this is not

clear.

In Table 4, it seems quite clear that the mobility of the hydro-

lyzed teichoic acid spots matched that of glycerol. The true picture,









however, is somewhat more cloudy than that. Anhydroribital is re-

ported to have the same mobility as glycerol under the conditions of

chromatography used in this experiment. Apparently, the conditions

of hydrolysis used for both the samples and the standard (2 N HC1,

1000C, 3 hours) were not sufficient to convert a detectable amount

of ribitol to anhydroribitol. Anhydroribitol is reported to give

a slow reaction with periodate-Schiff reagent (7) and so the fast

reaction given by the hydrolyzed teichoic acid samples confirms that

glycerol, not anhydrcribitol, was present. The yellow spots which

did not migrate were probably sugars which are characterized in

Table 4.

Both GS and LS crude teichoic acids appear to have the same sugars

present as evidenced by the banding patterns obtained in the ethyl

aceta te-pyridine-water solvent sys tem. Both preparations had a

silver nitrate reactive spot which migrated the same distance as

glucose. Both preparations also had darker areas in the long tails

which extended from the origin to about halfway to the solvent front.

These darker areas corresponded well to glucosamine and galactosamine

standards. Later analyses confirmed the covalent linkage of these

latter sugars to teichoic acid. The glucose is not connected with

teichnoic acid but may be part of the wall nevertheless.

Since the spots that were presumably glucose did not exactly

correspond in mobility to glucose in every run, glucose was added as

an internal standard to the GS and LS acid-hydrolyzed preparations

(Table 6). In this chromatograph standard glucose and sample glucose










migrated as a single spot. These spots moved slower than the glucose

standard alone. The probable cause for this is the presence of

small amounts of salt in the sample.



AImino Acid and kmnino Suagir_ Analyses of Teichoic Acid



Amino acid and amino sugar analyses confirmed the identity of the

glucosamine and galactosamine indicated by paper chromatography.

These analyses also indicated that there was about five times as

much glucosamine present as galactosamine.

The traces of alanine present would contribute less than one

residue for every 100 glycerol phosphate residues. For this reason,

it is felt that this alanine probably arose from contamination by

peptidoglycan.



DEAE Chromatography



To purify the crude teichoic acids, ion exchange chromatography

was used. The teichoic acid preparations were applied to the DEAE-

Sephadex under conditions which allowed the anionic teichoic acids

to stick to the resin. Water washes removed a similar amount of

phenal-sulfuric acid reactive material from both preparations. This

noaterial is presumed to contain the glucose present in crude teichoic

acid samples as demonstrated by paper chromatography. Lyophilization

of this material rendered it water-insoluble and so no further analyses

of this material were carried out. No phenol-H2S0q reactive material









was found in the salt-eluted fractions, so glucose is presumed not

to be part of the teichoic acid structure.

The profiles of Figure 3 show that GSTA and LSTA are different

in their affinity for DEAE groups. The peaks of phosphate eluted

at salt concentrations which were about 0.1 M~ different. This suggests

that there may be a difference in net charge of the molecules being

separated. These profiles also show that the amino sugar curve

closely matched that of phosphate. If the ratios between phosphate

and amino sugar are calculated for each fraction in the main peaks,

these ratios are relatively constant. They are so constant that in

more than 95% of the cases, the differences can be attributed to a

change from the mean ratio of an amount that can be accounted for

by a change of one or two residues. Thus suggests that these two

moieties were associated with each other and not simply eluting close

to one another.

Dry weight recoveries were quite low as compared to phosphate re-

coveries. This is probably due more to inaccuracies in dry weight

measurement (resulting from incomplete water removal from samples)

than to a removal of contamination.



Acid Hydrolyses of Purified Teichoic Acids



The amount of amino sugar in the two preparations was different

but not radically so. The mild acid hydrolysis only demonstrated

that about one half of the amino sugars were N-acetylated, but this

is strictly a minimum figure. Even under these mild conditions some

hydrolysis of the N-acety1 groups probably occurs. It is therefore








probable that most amina amino sugar was N-acetylated. DMAB reagent

showed a limited reactivity to amino sugar associated with teichoic

acid because the N-acetyl amino sugar is bound at the C-1 position.

N-Acetylation with acetic anhydride did not increase this reactivity.

This would seem to indicate that all the amino sugars are already

N-acetylated. When the teichoic acid was acid hydrolysed a large in-

crease in reactivity to DMAB occurred due to exposure of C-1.



Gel Filtration



Teichoic acids were also characterized by gel filtration. It

was found necessary to use Sephadex G-100, a gel with a molecular

weight exclusion limit much higher than any known teichoic acid

molecular weight. This is probably due to the high charge and ex-

tended shape common to teichoic acids (25,27). The profiles in

Figure 4 again show that there was a considerable difference between

GSTA and LSTA. The most obvious cause for this is a difference in

molecular weight but if there were a large difference in charge or

shape of the molecule, it could affect the retention of the teichoic

acid molecules.



Determination of Chain Length



To further characterize the teichoic acids and to determine if

molecular weight was the determining factor in the gel filtration

profile, chain lengths were determined. Average chain length can

be determined by releasing the terminal phosphomonoester group








with alkaline phosphatase and measuring the inorganic phosphate released

and comparing that to the total phosphate content. This gives the

average number of glycerol phosphate residues per chain if there

is a one-to-one correspondence of glycerol to phosphate. These deter-

minations showed that GSTA at 38 glycerol phosphate units was only

about half as long as LSTA at an average of 70 units. These numbers

are representative of purified teichoic acid preparations only and

may not reflect accurately the native chain lengths as found in the

wall (8). TCA is known to break phosphodiester bonds (77). Phos-

phodiester bonds not only link the glycerol phosphate monomers, they

are also responsible for linking the teichoic acid to peptidoglycan

in the only well-characterized cases to date (22,38,77). It is

not surprising then that a certain amount of degradation of chain

length would occur upon TCA extraction of teichoic acids. Indeed,

this has been observed in several cases (8,34,51). The kinetic data

seem to indicate that there may be two mechanisms of extraction.

This may be reflective of the phosphodiester links which bond the

backbone units and those which may link the backbone to peptidoglycan.

Even so, the difference between GSTA~ and LSTA would be hard to explain

by differing liabilities to acid extraction when their composition

is so similar. Thus, even if the chain lengths do not accurately

reflect those in the native wall, there still should be a significant

difference between the lengths of GSTA and LSTA in the wall.

It is very probable that the differing chain lengths of GSTA4

and LSTA account for some of the differences observed in ion-exchange

chr oma togr aphy. DEAE chromatography has been shown to separate

oligonucleotides according to net negative charge which is a function









of chain length under conditions minimizing charge and secondary

binding forces of the purine and pyrimidine bases (72,81,85). It

is reasonable to assume then that LSTA with an average of 70 negative

charges per molecule is going to be eluted at a higher ionic strength

than is GSTAl which only has as average of 38 negative charges per

molecule. This is, in fact, what is observed (Figure 3). This

reasoning can be extended to the material which elutes at lower

ionic strengths which would be of lower chain length than the bulk

of the material. This material probably resulted from degradation by

TCA hydrolysis of backbone phosphodiester bonds.

The difference in chain length also is reflected in the gel

filtration profile but it should be noted that the difference in

charge per molecule may also play a role in the behavior of teichoic

acids on Sephadex G-100. Pharmacia states that Sephadex G-100 contains

small numbers of carboxyl groups and, therefore, at low ionic strengths,

negatively charged moleclues may be excluded from the ge1 to a

greater extent than an uncharged molecule of the same molecular weight

(67).



Glycerol-Phosphate Ratios


Many of the above conclusions can only be drawn by assuming that

there is a poly (glycerol phosphate) backbone. To confirm the presence

of such a backbone, it is necessary to show a one-to-one ratio

between glycerol and phosphate. As Table 8 shows, this is the case

for both GSTA~ and LSTA.









Alkaline Hydrolysis


The final characterization of teichoic acid was by alkaline hydro-

lysis. The primary goal of this experiment was to establish whether

or not the amino sugars were covalently attached to teichoic acid.

Alkaline hydrolysis leaves glycosidic linkages intact while cleaving

phosphodiester linkages where free hydroxyl groups are available on

neighboring carbons allowing for a cyclic phosphate intermediate to

form (50) .

Figure 5 shows that a substantial amount of amino sugar elutes

from DEAE~-Sephadex as a single peak. Since only anionic molecules

should be bound to this resin, this suggests tha the amino sugar is

still bound to glycerol phosphate. This peak accounts for 87% of

the amino sugar in the alkali-hydrolyzed GSTA. Thus, the majority

of amino sugar is covalently attached to teichoic acid and it sug-

gests that the poly (glycerol phosphate) backbone is 2,3 linked rather

than 1,3 linked. In a 1,3 teichoic acid if a glycosidic bond ties
up C-2 of a glycrlpopaersde then there is no free hydroxyl

on that residue for the necessary cyclic phosphate to form. For this

reason, one expects only glycosylglycerol for substituted residues.

In contrast, glycosylglyceromonophophates can only result from a 2,3-

linked teichoic acid structure. It has never been found in membrane

teichoic acids and only in a few cases in wall teichoic acids (32,63).

The amount of amino sugar exceeds the amount of phosphate in the amino

sugar peak by a factor of about 3.2. This suggests that there may

be an average of three amino sugars linked to each other by








glycosidic bonds and this trimer is attached to a single glycerol

phosphate residue.

The other 12% of the amino sugar in the alkaline-hydrolyzed

GSTA was characterized as being low molecular weight because it was

almost completely included in a Sephadex G-50 gel (Figure 6) This

material was then eluted on a cation exchange resin. All the

remaining phosphate was washed through the column with 0.1 M NaCL.

Two peaks of amino sugar were then eluted using a linear N~aC1 gradient.

Together these accounted for all of the amino sugar from the DEAE

wash fraction. No phosphate was detected in either fraction. Bath

peaks did contain glycerol. The smaller peak contained about three

amino sugar residues for each glycerol residue while the larger

peak contained 6.5 amino sugars per glycerol. It would seem, there-

f~ore, that CI-Sephadex acts in manner analogous to DEAE-Sephadex in

that it can separate positively charged molecules with similar charge

densities by chain length. These data also suggest that amino sugar

is covalently attached to teichoic acid and may exist as trimers or

taxamers attached to single glycerol residues. The significance of

the larger amount of the hexamer in this relatively small fraction

of amino sugar is not known. Although these results were obtained

only for GSTA, it seems reasonable that LSTA follows a similar but

not identical pattern.





Summary of Teichoic Acid Structure



Arthrobaover cryistatlopoietes contains a glycerol wall teichoic

acid in both sphere and rod morphologies in approximately equal

amounts. This teichoic acid is 38 glycerol phosphate units long

in spheres and about 70 units long in rods. N-Acetylhexosamine

(N-acetylglucosamine about 83% and N-acetylgalactosamine, about 17%)

is attached by a glycosidic linkage from C1 of the hexosamine to Cl

of glycerol. On the average, there are 11 hexosamines per chain in

sphere teichoic acid while there are about 15 hexosanines per chain

in the rod teichoic acid. These hexosamines may exist in polymerized

side chains which have three sugars on the average. Thus, only about

11% (GS) or 7% (LS) of the glycerol phosphate units are substituted.

The poly (glycerol phosphate) backbone is 2,3 linked for GSTA and

probably for LSTA also. This is the first teichoic acid that has

been characterized to this extent in the genus Arthrobacter.



Possible Roles for Teichoic Acids in Alrtihrobacter



One role of teichoic acids that is well established is binding of

cations, particularly divalent cations (6,10,26,42,46). Evidence

has been presented that wall and membrane teichoic acids provide a

controlled reservoir of bound Mg+2 formembrane bound enzymes (46)

which require it for maximnal activity (2,21,66). It has been suggested

that the wall teichoic acid scavenges MIg+2 from the environment and

transfers it to lipoteichoic acid which interacts with the membrane








enzymes. Baddiley et al. (10) have shown that alanylated teichoic

acid interacts less strongly with MIg+2 and, therefore, the degree of

D-alanine substitution may act as a controlling influence on Mg+2

binding.

It seems reasonable that the ceichoic acid in A. cystailopoietes

could serve the same function since its structure is similar to

some of those used in the above mentioned studies. If this were

true, then it could be that the longer chains found in rod shaped

bacteria might more effectively scavenge Mg+ than the short chains

of the spheres. This would be consistent with the higher growth

rate of rods which would presumably necessitate a greater flux of

E + to the membrane.

Another function of teichoic acids is suggested by the studies

of Burge et al. (20) and Milward and Reavely (62). By examination of

peptidoglycans containing teichoic acid and those which had teichoic

acid artificially removed, these authors found that extraction of

teichoic acid affects the flexibility of the peptidoglycan. It was

suggested (20) that since teichoic acid can pass through the pores

in the outer layers of peptidoglycan it would resist shear between

adjacent peptidoglycan sheets and still allow for expansion or

contraction of the wall.

The most interesting function as far as this work is concerned

is suggested by several studies in which teichoic acid changes

effect autolytic enzymes.

Holtj e and Tiomas z (45, 80) have d described an N-ace tylImuramyl-L-alanine

amidase of Pneumnococcucs which required choline residues in the wall

teichoic acid for maximnal binding. If ethanolamine residues were









substituted, the cell walls were resistant to the autolysin. Besides

lowered binding of the autolysin to the wall, this effect was due to

the inability of the altered teichoic acid to convert the autolysin to

its active form. Herbold and Glaser (43) have described a similar N-

acetylmuramyl-L-alanine amidase in Bacillus subtilis. Again cell walls

lacking teichoic acid had a much lower affinity for the enzyme than

those with normal levels of teichoic acid. In a different strain of

3ac~ills subltilis, Brown at al. (16,18) had such trouble purifying auto-

lysin from teichoic acid that they suggested that the two molecules

might be covalently attached to each other. They speculated that the

close association between autolysin and the teichoic acid might serve

to localize the autolysin for maximum utilization of substrate.

In a temperature conditional morphological mutant of Baciilus

subtilis, Boylan et al. (12,13,24) found that at the non-permissive

temperature teichoic acid was deficient. These mutants grew as normal

rods at 300C but changed to irregular spheres at 450C. The walls of the

spheres were also deficient in N-acyl muramyl-L-alanine amidase. Rogers

et al. have found similar results in seven rod A4 mutants of BaoitZus

subitiis (17,71) and Baci'ius isickzen~iforis (33) .

Other authors (44,75) have also suggested that autolysins may

play a significant role in the determination of morphology in all

bacteria.

In A. crystalloDPOietes it has been shown that the activity of

:;-acetylmuramidase correlates with morphogenesis (53). The high

activity of the autolysin in spheres results in the relatively short

glycan backbones found in the peptidoglycan. In the same way, the

low activity of this enzyme in rod walls results in relatively





long glycans. The length of the glycan chains probably influences

the rigidity of the peptidoglycan and, therefore, the morphology

of the organism (55).

The present work has shown that there is almost a two-fold differ-

ence in the length of teichoic acids isolated from spherical and

rod-shaped A. crystaZlopoietes. In light of the correlations

between morphology, autolysins, and teichoic acids discussed above,

it does not seem unreasonable that this difference may play a role

in morphogenesis.

Phosphate analyses of purified wall show that there is slightly

more phosphate per mg of rod wall than in sphere wall (rods, 21 iug/ml

wall; spheres, 18 ug/mg wall). Since the rod teichoic acid has

almost twice the phosphate per chain as the sphere teichoic acid

because of its length, this means that there are about one half as

many rod teichoic acid chains as there are sphere teichoic acid

chains. If autolysin were binding to the teichoic acid chains, the

rod wall would present about one half as many binding sites proximal

to the wall as would the sphere wall.

A question that arises from this model is why the autolysin does

not cleave the ?J-acetylmuramyl-N-acetylglucosaminyl residue in peptido-

glycan to which it is attached. This would be disadvantageous to the

cell because both teichoic acid and autolysin would be lost from the

cell which represents a considerable energy investment wasted. One

possible solution to this problem could lie in the specificity of the

aurolysin. Thie autolysin in A?. crystailopoistes thought to be

responsible for control of glycan length is an N-acetylmuramidase.

It is possible that this enzyme can not hydrolyze at the reducing









end of an N-acetylmuramic acid which has the C-6 hydroxyl tied up in

covalent linkage. Lysozyme is known to be inhibited under these

conditions and teichoic acids are known to inhibit lysozyme in certain

cases (78). The only well characterized linkages of teichoic acid

to peptidoglycan are to the C-6 hydroxyl of N-acetylmuramic acid.

In both our laboratory and that of Krulwich et al. (55), lysozyme

was found to be poorly lytic against purified cell walls of A. cry~sta7,Zo-

poietes. Calaropjsis B enzyme, on the other hand, completely solubilizes

these walls. ,mChaLzropsis B enzyme is an N,0O-diacetylmuramidase

which is similar to lysozyme except that it will cleave at the reducing

ends of N-acetylmuramic acids which are substituted in the C-6 position.

So, apparently something is attached at this position in A. crystallo-

podetes. It is possible that this might be teichoic acid but it

could also be polysaccharide. If it were teichoic acid, it may be

the reason why autolysin does not remove teichoic acid. Krulwich

and Ensign (53) did find that the autolysin preferentially solubilizes

peptidoglycan which is low in phosphate content. Another reason for

this result might be steric hindrance of the enzyme by che teichoic

acid.

It may be that teichoic acid does not bind the autolysin at all

but only serves in substrate recognition by the enzyme as suggested
by Higgins and Shockman (4d). In Sreptococcus fa~ecais, the N-acetyl-

mnuramidase binds to TCA extracted walls but these walls are hydrolyzed

slower by autolysin than untreated walls. Th~e TCA extracted walls are,

however, a becter substrate for lysozyme.




73




It is also possible that the divalent cation binding property of

t-eichoic acids is important in regulating the autolysin. Clearly,

there is much more to be found out about the roles of teichoic acids

in A. crystalloPoietes. Hopefully, further research will shed light

on these roles.















LITERAZTURE CITED


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BIOGRAPHICAL, SKETCH


John H. Hellmuth was born September 3, 1952, in Jacksonville,

Florida. There he attended The Balles School and graduated in

iayI, 1970. In December, 1973, he received the degree of Bachelor

of Science with honors, with a major in biological sciences with

an emphasis in Microbiology from North Carolina State. From January,

1974, until the present time, he has pursued his work toward

the degree of Doctor of Philosophy in the Department of Microbiology

and Cell Science, University of Florida.

John H. Hellmuth was married August 25, 1973,to the former

Mlartha Jean Payne of Jacksonville, Florida, and is the father of

a daughter, Kelly Leigh.











































































il~.l-- ili-U -^-. *- I- ~' '--i


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.




Edward P. Previc, Chairman
Associate Professor of Microbiology
and Cell Science



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.




Arnold S. Bleiweis
Professor of Microbiology and
Cell Science



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.






Professor of Soil Science















I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.





Lonnie 0. Ingram
Associate Professor of Microbiology
and Cell Science





This dissertation was submitted to the G~raduate Faculty of the
Department of Microbiology and Cell Science in the College of Liberal
Arts and Sciences and to the Graduate Council, and was accepted
as partial fulfillment of the requirements for the degree of
Doctor of Philosoph~y.

December 1978





den, Graduate School




Full Text

PAGE 1

CHARACTERIZATION OF WALL TZICHOIC ACIDS IN TWO MORPHOLOGICAL FORMS OF Artkpobactev arvstallovoietes By JOHN HARDIN HELLMJTH A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1978

PAGE 2

ACKNOWLEDGEMENTS The author wishes to thank sincerely Dr. Edward Previc for his encouragement, suggestions, and criticisms during the preparation of this dissertation. He also wishes to thank the other members of his supervisory committee, Dr. Arnold Bleiweis, Dr. David Hubbell and Dr. Lonnie Ingram for the help they contributed in the preparation of this manuscript. The author would like to express his gratitude to the other members of the Department of Microbiology and Cell Science for generously supplying equipment and materials during his work. Special thanks to Steven Hurst for technical assistance with amino acid analyses. Appreciation is extended by the author to his parents for the encouragement they gave him to pursue his education. Finally, the author is particularly indebted to his wife, Martha, for patience, encouragement, and assistance during his graduate work.

PAGE 3

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES v LIST OF FIGURES vi ABSTRACT vii INTRODUCTION 1 Taxonomy 1 Control of Morphogenesis 3 Justification for This Study 5 Teichoic Acid Background 6 MATERIALS AND METHODS 9 Organism 9 Media 9 Growth 10 Cell Wall Isolation 10 Purification of Cell Wall 11 Teichoic Acid Isolation 12 Analysis of Purified Walls 12 Paper Chromatography 13 Analytical Procedures 14 Amino Acid and Amino Sugar Analysis 16 Teichoic Acid Purification 17 Gel Filtration 17 Chain Length Determination 18 Determination of Molar Ratios of Glycerol and Phosphate in Purified Teichoic Acids 19 Characterization of Alkaline Hydrolysate 19 RESULTS 22 Growth of ArtkrobaoteT crystallopoietes ATCC 15481 .... 22 Amino Acid and Amino Sugar Analysis of Purified Wall Hydrolysates 22 TCA Extraction of Purified Walls 25 Recoveries of Purified Wall and TCA Extractable Material (Crude Teichoic Acid) 29 Characterization of Acid-Hydrolyzed TCA Extracts by Paper Chromatography 29

PAGE 4

Page Amino Acid and Amino Sugar Analysis of Crude Teichoic Acid 35 Crude Teichoic Acid Purification on DEAE-Sephadex .... 35 Purified Teichoic Acid Characterization on Sephadex G-100 39 Chain Lenght Determination 39 Molar Ratios of Glycerol and Phosphate in Purified Teichoic Acids 43 Characterization of Alkaline Hydrolysates 43 DISCUSSION 52 Peptidoglycan Composition 52 TCA Extraction 57 Characterization of Acid Hydrolysates 59 Amino Acid and Amino Sugar Analyses of Teichoic Acid ... 61 DEAE Chromatography 61 Acid Hydrolyses of Purified Teichoic Acids 62 Gel Filtration 63 Determination of Chain Lenght 63 Glycerol-Phosphate Ratios 65 Alkaline Hydrolysis 66 Summary of Teichoic Acid Structure 68 Possible Roles for Teichoic Acids in Arthvobastev .... 68 LITERATURE CITED 74 BIOGRAPHICAL SKETCH 81

PAGE 5

LIST OF TABLES Table Page 1 Carbohydrate constituents of representative teichoic acids 8 2 Molar ratios of amino acids and amino sugars in walls digested with various enzymes 26 3 Paper chromatography of acid-hydrolyzed TCA extracts. I. Phosphoric acid esters 31 4 Paper chromatography of acid-hydrolyzed TCA extracts. II. cs-Glycols 32 5 Paper chromatography of acid-hydrolyzed TCA extracts. III. Sugars (external standard) 33 6 Paper chromatography of acid-hydrolyzed TCA extracts. IV. Sugars (internal standard) 34 Molar ratios of labile and total phosphate in alkaline phosphatase-digested, purified teichoic acids 42 8 Molar ratios of glycerol to inorganic phosphate .... 44

PAGE 6

LIST OF FIGURES Figure Page 1 Growth of Arthrobaater crystallopoietes 24 2 TCA extraction of purified walls 28 3 DEAE-Sephadex chromatography of crude teichoic acid 38 4 Sephadex G-100 chromatography of purified teichoic acid 41 5 Alkali-hydrolyzed teichoic acid elution on DEAESephadex 47 6 Sephadex G-50 chromatography of neutral and cationic components of alkali-hydrolyzed GSTA 49 7 Ot-Sephadex chromatography of neutral and cationic components of alkali-hydrolyzed GSTA 51

PAGE 7

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF WALL TEICHOIC ACIDS IN TWO MORPHOLOGICAL FORMS OF Art hrobacter orvstallovaietes 3y John Hardin Hellmuth December 1978 Chairman: Dr. Edward P. Previc Major Department: Microbiology and Cell Science The cell wall teichoic acid isolated from two morphological forms of Art Ivobacier crystallopoistes is characterized. Cell walls purified from spherical (GS) cells contained 13.2 ug of phosphorus per mg of cell wall, while those from rod-shaped (LS) cells contained 21.2 ug phosphorus per rag of cell wall. Trichloroacetic acid extracts of purified walls of both forms were found to contain poly (glycerol phosphate) with hexosamine glycosidically attached. In GS teichoic acid there was 2S% as much hexosamine as glycerol phosphate and in LS teichoic acid there was 21% as much. The hexosamine included at least 50% N-acetylated glucosamine and gaiactosamine in about a 5-to-l ratio. Evidence is presented which suggests that the hexosamine may exist as trisaccharide side chains. Chain lengths were estimated by the racic of total phosphate to alkaline phcsphatase-labile phosphate. By this raechcd, teichoic acids from GS-grown cells had an average length of 33 glycerol phosphate

PAGE 8

units and those from LS-grown cells had an average length of 70 units. The possible significance ot these findings as they relate to morphogenesis in A.rt hpobaotev is discussed.

PAGE 9

INTRODUCTION Taxonomy The genus Arthrobaater is characterized by cells which can undergo nutritionally-controlled sphere-rod morphogenesis (43). The species Arthrobacve? ovysialloyoietes was first described by Ensign and Rittenberg (30) who isolated the organism by enrichment cultures containing 2-pyridone. The brilliant green crystalline pigment produced by this species growing on solid medium containing 2-pyridone was later identified by Kuhn et al. (57) to be a hydrate of the monopotassium salt of 4,5,4' ,5'-tetrahydroxy-3,3'-diazadiphenoquinone(2,2'). Recently, Kolenbrander and Weinberger (52) found that A. arystallopoietes lost the ability to produce pigment from 2-pyridone at a high spontaneous frequency of 0.26% loss per generation. This high spontaneous loss has also been observed in the strain of A. evystallovo'i.etes used in this dissertation research. Kolenbrander and Weinberger (52) present good evidence that the loss of a plasmid is correlated with loss of ability to produce pigment. In the present study this presented a problem since the plasmid-less strain seemed to grow faster than the parent strain in glucose-salts media. It was therefore necessary to minimize the number of accumulated mutants by starting each culture for harvest from a single pigment producing colony.

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According to Bergey's Manual (48), the name Arthrobaotev ovystallopoietes is a probable subjective synonym of Arthrobaoter globiformis. The Manual also states that the salient feature distinguishing A. crystallopoietes from .4. globiformis is the ability to utilize 2-pyridone as a sole carbon and energy source and to produce a crystalline pigment from it. It thus seems likely that A. cvystallopoietes which has lost the plasmid has almost exactly the same phenotype as A. globiformis. Since the presence or absence of the plasmid may affect the wall composition by either direct effects (e.g., plasmid genes might modify wall synthesis) or indirect effects (e.g., plasmid presence might affect growth rate which might in turn affect wall synthesis) , comparisons between the walls of these bacteria can not be made on the assumption that A., cvystallovoietes and A. globiformis are the same bacteria. The nutritional control of morphology in A. cvystallopoietes was first demonstrated by Ensign and Wolfe (31). They showed that exponential growth of spheres could be obtained in a defined medium containing glucose. Exponential growth of rods could be obtained by adding certain morphogenesis-inducing compounds to the defined glucose medium. This idea forms the basis for the methods of obtaining spheres and rods in the following work. The only difference is that the rodinducing medium contains lactate but no glucose. Since there is a diauxic suppression of glucose cataboiism and anabolism in the presence of rod-inducing compounds (54), this difference is minimized.

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Control of Morphogenesis Although the morphology of Arthrobaeter can be extrinsically controlled by nutritional means, this control is only indirectly related to the unknown intrinsic control by the bacteria. The intrinsic control is more directly related to growth rate which can be controlled independently from the type of nutrition. Luscombe and Gray (60) have done this by growing a strain of Arthrobaater under carbon-limiting conditions in a chemostat. They found that, at 25°C, rods were only produced at dilution rates above 0.25 per hour. At rates lower than this, the cells were always spherical. This raises a problem because it means that whenever rod and sphere-shaped arthrobacters are compared, morphology is not the only difference; growth rate also changes and hence could affect many variables. This is a fundamental problem which makes it difficult to establish correlations as causal relationships and it applies to this study. Hamilton et al. (39) have sidestepped this problem by isolating a spherical morphological mutant of A. crystallopoietes which is unable to undergo sphere-rod morphogenesis but increases its growth rate in rod-inducing media. The genetic nature of this lesion has not yet been reported. The intrinsic control of morphogenesis probably consists of a chain of events, some of which are genetic, some enzymatic, and some structural. Certain changes at any level of this chain can probably manifest themselves at the morphological level. Therefore, any one event should not be said to control morphogenesis. Nevertheless, several authors (39,47,53,61,68,69,73) claim that their effect which

PAGE 12

correlates with morphology is probably the factor which controls morphogenesis in .4. arystallopoietes . It has been shown that A. arystallopoietes contains two RNA polymerases both of which have considerable and almost equal synthetic capabilities in in vitro studies (47). This correlates with another finding by the same lab (61) that morphogenesis involves differential transcription of the DNA with some transcripts being present all the time and others being present only at certain times during the morphogenetic cycle. Furthermore, St. John and Ensign (73) by using RNA and DNA synthesis inhibitors were able to show that morphogenesis can occur in the absence of DNA replication but RNA synthesis is required for morphogenesis to occur. Hamilton et al. (39) suggest that the level of cyclic adenosine 3' ,5 '-monophosphate (c-AMP) may be important in regulating morphogenesis. The levels of c-AMP were shown to rise steeply at times just prior to the initiation of shape changes (either sphere-to-rod or rodto-sphere). This suggested that elevated c-AMP levels may act as the "trigger" which induces the cells to change shape. Their morphological mutant (mentioned above) did not show these changes. Krulwich et al. (53, 55) have shown that the glycans in the peptidoglycan of spheres are generally shorter than those of rods. This change was also correlated with a change in the activity of a wallbound N-acetylmuramidase. They implied that morphology, therefore, depends on glycan chain length. Previc (68) has suggested that morphology in most bacteria may be determined by the presence or absence of extra crossbridges involving free carboxly groups of diaminopimelic acid (Dpm) and other

PAGE 13

tetrafunctional amino acid groupings (e.g., lysylaspartyl crossbridges in some Lactobacillus species). In a mutant of A. cv^jstallovoietes Previc and Lowell (69) have shown that spherical mutants contain lysine in the penultimate position of tetrapeptides while rod-shaped mutants contain Dpm. Transitional stages during sphere-to-rod morphogenesis show a gradually increasing amount of Dpm present. Thus, morphology in this strain may be dictated by the presence or absence of the extra carboxyl group of Dpm. Several authors seem to agree that a rod shaped morphology requires a more rigid peptidoglycan than a spherical morphology (37,55,68). Ward and Claus (84) have found that for .4. cvystallovoistes the rod peptidoglycan layer is thinner than the sphere peptidoglycan. If the rod peptidoglycan is more rigid than the sphere peptidoglycan, then this difference must be due to changes in the peptidoglycan structure rather than simply a thickening of the wall. It should now be apparent that there are changes during morphogenesis at each step in the chain of events which regulates morphology. What is still not apparent is how all these changes fit together to produce morphogenesis. Of course, some of the changes may not be related to morphogenesis at all but only reflect altered growth rates. So, the question of how morphogenesis occurs is still unanswered. Justification for This Study The original intent of this author was to determine how peptidoglycan might help to maintain the different shapes of A. arystalloyoietes. To accomplish this the composition of the wall was examined for changes

PAGE 14

which might correlate with morphology. During the beginning of the quantitative analyses of the components of the wall, there arose certain evidence that a wall teichoic acid might be present. The determination of its qualitative and quantitative structure has become the primary topic of this dissertation. Although this seems far removed from the question of morphogenesis, there may be an intimate association between the two. These possibilities are examined in the Discussion. Teichoic Acid Background Teichoic acids are molecules which occur in nearly all gram-positive bacteria (3). These molecules are grouped into two categories depending on their cellular location and their structure; lipoteichoic acids are found associated with the cell membrane and wall teichoic acids are associated with the peptidoglycan (4) . Lipoteichoic acids are found in most gram-positive bacteria. These molecules are all of the same structural type, i.e., they consist of a linear backbone of poly (glycerol phosphate) which is linked by phosphodiester bonds involving C-l and C-3 of adjacent glycerol phosphates (3). Diversity of lipoteichoic acid structure is introduced by various carbohydrate side groups (see Table 1) which are attached at the C-2 hydroxyl group. D-Alanine is usually found as an ester linked either to the C-2 hydroxyl of glycerol phosphate or to glycosyl hydroxyl groups. It is though that all lipoteichoic acids are covalently attached to glycol.ipid in the cell membrane (51) .

PAGE 15

Wall teichoic acids are more structurally diverse than lipoteichoic acids. The classical wall teichoic acids are polymers of glycerol or ribitol phosphate. Both types can have various carbohydrate side groups (see Table 1) and/or D-alanine. These polymers are thought to be covalently attached to the peptidoglycan in the cell wall. Other acidic polymers have been found in the walls of grampositive bacteria which resemble the classical teichoic acids. Polymers of glycerol phosphate and one or more sugars have been found in a number of cases and polymers of sugar phosphates are also known. Both types of polymers are similar to teichoic acids in that they convey a net negative charge to the outer surface of the cell and, therefore, may have functions similar to the teichoic acids (51).

PAGE 16

r-x

PAGE 17

MATERIALS AND METHODS Organism Arthrobactar arystallopoietes ATCC 15481 was obtained from the American Type Culture Collection. In order to maintain the presence of the plasmid which bears the gene(s) responsible for green crystal production from 2-pyridone, stock cultures were frequently streaked on 2-pyridone-containing plates (30) and pigmented colonies were picked for further subculturing. All inocula for cultures to be harvested were also checked in this manner. Media Defined media contained, per liter: 1.73 g K 2 HP0 4 , 2.33 g KH 2 P0 4 , 1.00 g (NH 4 ) 2 S04, 5.0 g D-glucose (GS) or sodium lactate (LS) , 0.5 g MgS0 4 , and 10.0 ml of a trace salts solution. The trace salts solution contained, per liter: 1.5 g nitriloacetic acid, 0.5 g MnS0 4 , 1.0 g NaCl; FeCl 2 , CaCl 2 , CoCl 2 , and ZnS0 4 , 0.1 g each, and CuS0 4 , A1K(S0 4 ) 2 , H3BO3, and (NH 4 ) 6 Mo 7 2 4, 0.01 g each. The trace salts solution was adjusted to pH 7.0 with 1.76 g NaOH. The glucose, MgSO, and the trace salts solutions were each autoclaved separately (31) A complex pigmentation medium containg 0.2% 2-pyridone, 0.05% yeast extract, and inorganic salts was prepared according to Ensign and Rittenberg (30).

PAGE 18

10 Growth All cultures were agitated at 30°C in a New Brunswick Gyrotory shaker-incubator. Optical density (O.D.) was followed at 450 nm on a Beckman DU-2 spectrophotometer with a 1.0 cm light path. Optical densities were maintained between 0.05 and 1.0 O.D. units by periodic transfers to fresh media. Cell Wall Isolation Exponential cultures were passed through coiled copper tubing which was submersed in an ice-water bath. The cooled effluent was directed into a De Laval Gyro Tester Laboratory Centrifuge with the bowl prechilled to 4°C. The time for culture liquid to pass through the cooling coils and out the centrifuge spout was measured using enough nigrosin dye added to a deionized water run to serve as a visual marker. The temperature and O.D. at 450 nm of the final effluent were also measured during bacterial harvests. From these measurements it was concluded that cultures were cooled from 30°C to 8-10°C in approximately one minute and that 95% or more of the culture's bacterial mass was removed from the effluent as determined by O.D. measurement. All subsequent handling of bacterial samples was at no higher than 4°C unless otherwise specified. Whole cell pellets were washed twice by resuspension in deionized water and centrifugation at 13,000 x g for 30 min in a Servall GSA rotor. Bacteria were suspended in deionized water at a concentration no greater than 50 mg dry wt/ml and broken in a Braun homogenizer at 4,000 rpm for 4 minutes using 0.11 mm

PAGE 19

II diameter glass beads. Tributyl phosphate (0.5% v/v) was used to reduce foaming. Glass beads were removed first by filtration on fritted glass filters and then by centrif ugation at 160 x g for 30 min. Crude cell walls were then pelleted from the supernatant at 27,000 x g for one hour. Purification of Cell Wall The method of Braun and Sieglin (14) for wall purification was modified by an additional sodium dodecyl sulfate (SDS) extraction before treatment with pronase (69) . Crude wall pellets were suspended in 0.1 M ethylenediaminetetraacetic acid (EDTA) , pH 7.4 (at least 20 ml/g of whole cell dry wt), and then pelleted at 27,000 x g, washed and resuspended in deionized water. Suspensions were added dropwise to stirred, boiling 4% SDS (at least 15 ml/g of whole cell dry wt) . These mixtures were allowed to cool to room temperature with stirring overnight. These mixtures were then centrif uged at 27,000 x g for one hour. Pellets were resuspended in deionized water and reextracted with boiling kl SDS. These pellets were washed with deionized water three times and resuspended in 0.05 M Tris, pH 7.4 (at least 5 ml/g of whole cell dry wt) . Pronase was added to a final concentration of 100 jig/ml and the mixtures were digested at 37°C for 16 hours. The walls were centrif uged for one hour at 37,000 x g. The resultant pellets were reextracted by boiling SDS as above and rewashed three times with deionized water. The final pellets were lyophilized for dry weight determinations.

PAGE 20

12 Teichoic Acid Isolation To release teichoic acid (G), purified wallswere extracted with 10% (w/v) tricloroacetic acid (TCA) at 4°C for 24 hours. Walls were pelleted from this mixture at 27,000 x g for one hour and the pellets were reextracted with fresh 10% TCA at 4°C for 24 hours. Again fresh cold TCA was exchanged for supernatant TCA and the extraction was continued for 38 hours. Crude teichoic acids were collected either by precipitation from the TCA with absolute ethanol (5 volumes) at -20°C and subsequent centrifugation at 13,000 x g for 30 minutes, or by removing the TCA by ether extraction to pH 4 and subsequent lyophilization. Analysis of Purified Walls Purified walls (0.4 mg/ml) were hydrolyzed in 4 N HC1 at 105°C for 11 hours. The excess acid was then removed by evaporation, followed by three cycles of deionized water additions and reevaporations. The residues were dissolved in 0.01 N HC1 (1 mg wall/ml acid) and then filtered through 0.45 um Millipore filters. Unsolubilized material was present in negligible amounts. The filtrates were analysed for amino acids and amino sugars on a JE0LC0 Automated Amino Acid Analyzer (76) .

PAGE 21

13 Paper Chromatography Whatman papers no. 1, no. 4, and no. 3 MM were soaked for at least 30 minutes in a solution which was 0.1 N in acetic acid and 0.1 M in EDTA. This solution was then washed out by suspending the papers in deionized water and then ramoving excess water by decantation. At least ten sequential water washes of increasing duration were performed, with a final wash of at least one hour. Washed papers were allowed to dry in horizontal stacks at 30°C. Papers to be eluted parallel to the grain of the paper were cut to 23 cm x 57 cm with the grain running parallel to the long axis of the paper. Papers to be eluted perpendicular to the grain were cut to 23 cm x 46 cm with the grain running perpendicular to the long axis. Samples were spotted 7 cm from the top of the papers by repeated application of approximately 0.25 ul each time, then drying by hot air. The papers were mountainfolded along a line at 6 cm and valley-folded along a line at 3 cm from the top of the papers. This folding allowed the papers to be hung from a glass trough in a pyrex tank (30.5 cm x 30.5 cm x 61 cm). The papers were allowed to equilibrate with the vapor phase of the eluting solvent in a glass chromatography tank for at least 2 hours before the eluting solvent was added for the beginning of elution. All chromatograms were eluted at ambient temperature, typically ranging from 22°-27°C. The solvent systems for elution used were: (A) n-propanolammonia (28-30%)-water (6:3:1) (40) and (3) ethyl acetate-pyridine-water (10:4:3) (36).

PAGE 22

Analytical Procedures Phosphate Assays Total phosphate was assayed by the ascorbic acid-molybdate method developed by Chen et al. (23). The ashing method of Lowry et al. (58) was used for convenience in handling large numbers of samples. Inorganic phosphate was measured by the Chen method as modified by Ames (1). KH2PO4 (anhydrous analytical reagent, Mallinckrodt) was used as a standard in both total and inorganic phosphate methods. Chromic acid-washed tubes were used for all phosphate assays. Amino Sugar Assays N-Acetylamino sugars were measured by the borate-p-dimethyl-aminobenzaldehyde (DMAB) method of Reissig et al. (70) . N-Acetylglucosamine (Sigma) was used as the standard for this assay. Amino sugars were assayed for N-acetylation with acetic anhydride by a method similar to the N-acetylamino sugar assay as described by Ghuysen et al. (35). This method primarily detects amino sugars which are free in the C-l position but it also detects amino sugars which are covalently bound at that position. The extinction coefficient for the latter reaction, however, is more than thirty fold smaller than that for the former reaction. D-Glucosamine-HCl (K+K Laboratories, Inc.) was used as a standard for this assay. Total amino sugars could be detected if an acid hydrolysis (2 N HC1, 100 C C, 3 hours) followed by neutralization preceded the N-acetylation step of the amino sugar assay. The same standard was used as for the amino sugar assay.

PAGE 23

15 The minimum amount of N-acetylation of amino sugar was estimated by a method suggested fay Ellwood et al . (29). This method involves a mild acid hydrolysis (0.1 N H 2 S0 4 , 100°C, 30 min in a sealed ampoule) which breaks the glycosidic linkage while leaving most of the N-acetyl groups intact. After hydrolysis, a slight excess (10%) of the amount of 3aC0 3 necessary for neutralization was added. The resulting i 3aS0 4 -BaC0 3 mixture was removed by low speed centrif ugation. N-Acetylhexosamines and total amino sugars were then determined for the neutralized samples. Carbohydrate Assay Simple sugars, oligosaccharides, and polysaccharides with either free or potentially free reducing groups were determined by the phenolH 2 S0 4 method of Dubois et al. (28). D-Glucose (analytical reagent, Mallinckrodt) was used as a standard. Protein Assay Protein was measured by the Lowry method (59) which involves first a reaction of protein with Cu +2 under alkaline conditions and then reduction of a phosphomolybdate-phosphotungstate reagent by the copper-treated protein. 3ovine serum albumin (Sigma) was used as a standard.

PAGE 24

16 Enzymatic Determination of Glycerol Free glycerol was determined by using the Glycerol Stat-Pack (Calbiochem) . This assay employs the following reaction sequence: (1) glycerol + adenosine triphosphate (ATP) glycerol kinase a-glycerophosphate + adenosine diphosphate (ADP) (2) ADP + phosphoenolpyruvate Pyruvate kinase ^ pyruvate + ATP (3) pyruvate + nicotinamide adenine dinucleotide (reduced) (NADH) lactate dehydrogenase i „„*„.„ . , . , , 2 _^ lactate + nicotinamide adenine dinucleotide (oxidized) The disappearance of NADH was followed spectrophotometrically at a wavelength of 340 nm using the same instrument used for O.D. readings. Glycerol (analytical reagent, Mallinckrodt) was used as a standard. Amino Acid and Amino Sugar Analysis Crude teichoic acid samples (GS, 5.0 mg; LS, 5.6 mg) were each hydrolysed in 1.0 ml of 2N HC1 at 100°C for 3 hours. The hydrolysates were vacuum dried over NaOH pellets and then redissolved in 1.0 ml of deionized water. Portions of these (200 yl each) were diluted in 0.01 N HC1 to 2.2 ml (final concentrations: GS , 0.45 mg/ml; LS, 0.51 mg/ml). These samples were subjected to amino acid and amino sugar analysis in the same way as described for purified wall hydrolysates.

PAGE 25

Teichoic Acid Purification Crude teichoic acids were purified on approximately 9 g of DEAESephadex A-50. One cm of Sephadex G-25 course gel was used at the bottom of the column as bed protection. The DEAE-Sephadex was swollen and loaded in 0.1 M NaCl. The bed diameter was 2.5 cm and the bed length varied depending on ionic strength (35.5 cm at 0.1 M NaCl and 24.0 cm at 1.0 M NaCl). The bed was then washed with 500 ml of 0.1 M NaCl. Teichoic acids were eluted by an increasing gradient of NaCl. For the LS preparation, 200 ml of a 0.1 M 0.5 M gradient was followed by a 200 ml gradient from 0.5 M to 1.0 M NaCl. A total of 160 fractions of approximately 2.1 ml each was collected. For the GS preparation, 500 ml of a 0.1 M 1.0 M NaCl gradient was used of elution. A total of 95 fractions of approximately 5 . 3 ml each was collected. The fraction numbers for the GS elution profile in the Results section have been normalized with the LS profile using equivalent salt concentrations . Gel Filtration Gel filtration was carried out on Sephadex G-100. Purified teichoic acids (GS, 42 mg; LS , 6 mg) were applied to a column bed of dimentions: 2.5 cm x 31 cm. Total bed volume was 160 ml. The void volume was determined to be 54 ml by using Blue Dextran 2000 (Pharmacia, Average molecular weight, 2 x 10 6 ) . A Blue Dextran sample (1 ml of a 0.2% solution) was applied to a column and the effluent was collected in

PAGE 26

approximately 3 ml fractions. These fractions were monitered for O.D. at 260 nm as a measure of Blue Dextran. The number of the fraction with the highest O.D. was multiplied by the fraction volume to obtain the void volume. Eluant was deionized water. A constant pressure head of 31 cm was maintained during loading and running. A Gilson automatic fraction collector was adjusted to collect 95 drop (approximately 3 ml) fractions. Results were graphed in terms of pmoles of total phosphate vs. K. K is defined as: V e V Q K = V t V c where: V = void volume V t = total bed volume Chain Length Determination In two separate determinations, about 1 mg and 0.5 mg of each type (GS and LS) of purified teichoic acid was diluted from concentrated solutions to 125 yl with deionized water. Then, 125 ul of 0.04 M (NH 4 ) 2 C03 and 10 pi (0.6 mg or 7.2 units) of Escherichia ooli alkaline phosphatase (60 mg/ml, 12 units/mg, Worthington Biochemical Corporation, Code: BAPSF bacterial alkaline phosphatase salt fraction) were added. One unit is defined as that activity liberating one umoie of p-nitrophenol per minute at pH 8.0 and 25°C. The mixture was incubated in a slowly shaking 37°C water bath for 18 hr. The samples were then analyzed for inorganic and total phosphate content. The ratio of total to inorganic phosphate content was taken as an approximation of chain length.

PAGE 27

19 Determination of Molar Ratios of Glycerol and Phosphate in Purified Teichoic Acids Purified teichoic acid samples were hydrolyzed in 2 N HC1 at 100°C for 4 hours. Acid was removed hy vacuum evaporation or lyophilization. The samples were then digested with alkaline phosphatase by the same general method described under chain length determination. The digested samples were then analysed for free glycerol, inorganic and total phosphorus. Because of phosphorus present in the enzyme preparation, it was also necessary to run enzyme blanks with no added sample and to subtract out the blank values from the sample values. This method was tested on a known concentration of ct-glyceromonophosphate which produced a molar ratio of glycerol to phosphate of 0.997. Characterization of Alkaline Hydrolysate A purified teichoic acid sample (GS, 32.6 umoles total phosphate) was hydrolyzed in 1.45 ml of 1 M NaOH at 100°C for 3 hours. The hydrolysate was neutralized with 300 ul of 4 N HC1. The final pH was close to pH 7.0 as judged by pH paper. The neutralized sample was diluted with deionized water to 14.5 ml to attain a salt concentration of 0.1 M NaCl. The diluted sample was loaded on the same DEAE-Sephadex A-50 column used for the teichoic acid puruf ication. In preparation for this run, the column had been previously washed with 500 ml of 1 M NaCl. The washed column was slowly reswollen with a 1.0 M 0.1 M

PAGE 28

mr NaCl gradient of 500 ml. Finally, the column was washed with another 500 ml of 0.1 M NaCl for final equilibration. Once loaded, the sample was washed with 500 ml of 0.1 M NaCl. The wash effluent was collected and lyophilized. The adherent portion of the sample was eluted from the column with 500 ml of a 0.1 M 1.0 M NaCl gradient. Fractions (100, approximately 4 ml each) were collected. These fractions were assayed for total phosphate and total amino sugars. The lyophilized wash sample was then filtered on a Sephadex G-50 column (diameter 1.6 cm, height 62 cm, 124 ml bed volume, 56 ml void volume) with deionized water in order to resolve low-molecular weight, non-anionic fragments. Fractions (65 of 3 ml each) were collected and analyzed for total and inorganic phosphate and total amino sugars. All phosphate-containing fractions were then pooled and lyophilized. This material (approximately 2.8 g) was assumed to be mostly NaCl and was diluted with deionized water to 0.1 M NaCl on that basis. This diluted sample was applied to a CM-Sephadex C-50 column (approximately 1.9 g of gel; column with following dimensions: diameter, 1.5 cm, length, 30 cm) to further resolve cationic molecules. The column bed was then washed with 50 ml of 0.1 M NaCl. After washing, the remaining sample was eluted with 250 ml of a 0.1 M 1.0 M NaCl gradient, and 46 fractions (5.4 ml each) were collected. These fractions were analyzed for total phosphorus and total amino sugars. The two peaks (two fractions/peak) containing significant amounts of hexosamine were each combined and lyophilized. These lyophilized samples were triturated in the presence of absolute

PAGE 29

ethanol (10) . The ethanol fractions were then evaporated to dryness and the residues were triturated again with absolute ethanol (1 ml) . The ethanol-soluble portions were again evaporated to dryness. The residues were each dissolved in 450 ul of deionized water and then analyzed for free glycerol.

PAGE 30

RESULTS Growth of Avilwobaatev ovy staltouo'istes ATCC 15481 Typical growth curves are shown in Figure 1. Generation times were: GS, 10.8 hr ± 0.5 hr; LS , 3.5 hr + 0.2 hr. Amino Acid and Amino Sugar Analysis of Purified Wall Hydrolysates It was observed that cell walls purified according to the protocol in Materials and Methods usually contained certain non-peptidoglycan amino acids. The following experiment was an attempt to remove these amino acids using proteolytic enzymes. Cell walls were isolated from lyophilized cells (GS or LS, 300 mg each) and were purified as described in Material and Methods with one modification. Just before the pronase digestion, each sample was divided into four equivalent portions. One portion of each was not treated with any enzyme and served as the control; the second portions were treated with pronase (500 ug/ml) ; the third portions were treated with trypsin (100 ug/ml); and the fourth portions were treated with ot-chymo trypsin (100 ug/ml) . All digestions were carried out in 0.05 M Tris, pH 7.4 at 37°C for 16 hours. Following the enzymatic digestions, each sample was treated as described in Materials and Methods for the rest of the wall purification and 22

PAGE 31

Figure 1. Growth of Art hrobaoter crystallopoietes. GS (7), LS (V).

PAGE 32

24 time, hours

PAGE 33

amino acid analysis. Peak areas on chromatograms were computed by a JOELCO integrater. Relative concentrations were then computed using known areas of standard amino acids and sugars. These relative concentrations were then divided by the relative concentrations for the glutamic acid peak on each chromatogram. The values for these ratios are given in Table 2. These analyses indicate that the normal peptidoglycan amino acids are present in the expected ratios. They also show that trypsin is the most effective enzyme in removing the trace amino acids. Chemical analysis of purified cell wall hydrolysates also shows the presence of significant amounts of phosphorus (GS, 18.2 tig P/mg wall; LS, 21.2 ug P/mg wall). Since Krulwich and Ensign (55) had reported phosphorus in their cell wall preparations of .4. ovystalloipoietes and had found that all of it could be removed by hot TCA extraction, a similar experiment was carried out with the cell walls prepared in this laboratory. TCA Extraction of Purified Walls Purified cell walls (GS, 19 mg; LS , 18 rag) were suspended in 10 ml of cold 10% TCA and were stirred with a magnetic stirring bar at 4 C. At various times, 1.0 ml portions were removed and extracted three times with petroleum ether (B.P. 30°C) to remove the TCA. The sample was then centrifuged at 27,000 x g for a half hour. Total phosphate was determined for both the soluble and insoluble fractions as shown in Figure 2. Phosphate was extracted by TCA with

PAGE 34

^^ TABLE 2 Molar ratios of amino acids and amino sugars in walls digested a with various enzymes.

PAGE 35

CO

PAGE 36

||dm Bui/6rt 'j

PAGE 37

time as indicated by the increase in TCA-soluble phosphate. This increase was paralleled by an equivalent decrease in wall associated phosphate as indicated by the TCA-insoluble curves. Recoveries of Purified Wall and TCA Extractable Material ( Crude Teichoic Acid ) Walls purified according to the scheme outlined in Material and Methods yielded 12-14% wall dry weight to cell dry weight for GS cells and 13-15% for LS cells. In each individual case the LS cells yielded slightly more wall (6-10% more) on a percentage basis than the GS cells. Recoveries of TCA-extractable material varied depending on whether ethanol precipitation or ether extraction was used to remove TCA from the samples. When ethanol precipitation was used, TCA extractable material recoveries (as crude TA dry weight percentage of purified wall dry weight) were: GS, 21%; LS , 18%. When ether extraction was used the recoveries were much higher: GS, 38% and LS, 28%. Characterization of Acid-Hydrolyzed TCA Extracts by Paper Chromatography To determine the nature of this phosphorus containing compound, acid hydrolyses were performed. The conditions used have been reported to degrade teichoic acids of either polyglycerophosphate or polyribitol phosphate and substituted with either amino acids or carbohydrate (3) .

PAGE 38

TCA extracts from two separate preparations were hydrolyzed in 2 N HC1 at 100°C for 3 hours. The residues were dried in vacuo over NaOH and then redissolved in deionized water. Approximately 25 ug of each sample was spotted on each of two washed Whatman #4 papers along with standards. The papers were then run with solvent system A perpendicular to the grain. One paper was run ascending for 32 hours. The other paper was run descending for 5 hours. Both papers were air dried. Phosphoric acid esters were detected by the acid-molybdate spray (40). The results of these chromatograms are given in Table 3. The unknown spots had mobilities characteristic of 1,2-diphosphoglycerol. Approximately 50 ug of hydrolyzed TCA extract were run under descending conditions in solvent system A but, in this case, ct-glycols were detected with periodate-Schif f spray reagent (9) . The results are given in Table 4. 3y detecting a-glycols the acid hydrolysate was shown to contain glycerol and not ribitol. The acid hydrolysates of teichoic acid were also examined for sugar content. About 50 yg of each sample was spotted on washed Whatman //l paper along with appropriate standards. The chromatogram was run with an ascending front of solvent system B for 18 hours. After air drying, sugars were detected by the silver nitrate-NaOH spray (19,82). The results are shown in Table 5. The unknowns in this case show mobilities characteristic of glucose and probably glucosamine. To confirm the identity of glucose in the unknowns, glucose was added to each unknown as an internal standard on similar chromatograms. The results are shown in Table 6. These results tend to confirm the presence of glucose in acid-hydrolyzed crude teichoic acids.

PAGE 39

TABLE 3 Paper chromatography of acidhydrolyzed TCA extracts. 2 I. Phosphoric acid esters. 31 Standards Ascending Descending 1,2 diphosphoglycerol (50 nmoles) a-glycerophosphate (100 nmoles) S-glycerophosphate (100 nmoles) hydrolyzed cardiolipin" (50 nmoles) Samples GSTA (25 yg) LSTA (25 yg) 0.14 L 0.09 C 0.24 0.31 0.13 0.09 0.14 0.10 0.28 0.16 C 0.39 0.39 0.35 0.12 0.11 0.14 (a) Elution was with solvent system A and spots were detected with the acid-molybdate spray reagent for phosphoric acid esters. (b) faint blue spot (c) dark blue spot (d) Hydrolysed cardiolipin was used for a 1,3-diphosphoglycerol standard. (e) not run

PAGE 40

3T TABLE 4 Paper chromatography of acid-hydrolyzed TCA extracts. 3 II. a-Glvcols Standards c a-glycerophosphate 0.42 (600 nmoles) hydrolyzed ribitol 0,55 (65 nmoles) glycerol 0.75 (1 umole) Samples GSTA (50 yg) .73 b LSTA (50 yg) 0>74 b (a) Elution was descending with solvent system A and spots were detected using the periodate-Schif f spray reagent for ct-glycols. (b) Also had yellow spots at the origin which were the only spots to develop color slowly.

PAGE 41

33 TABLE 5 a Paper chromatography of acid-hydrolyzed TCA extracts III. Sugars (external standards). Standards R , b glucose Glucose 1.00 (5 ug) Galactose 0.88 (5 ug) Glucosamine 0.39 (5 ug) Galactosamine 0.30 (5 ug) Samples GSTA (50 ug) 1.01 0.53 C LSTA (50 ug) 1.10 0.53 C (a) Elution was ascending with solvent system B and spots were detected by the silver nitrate-NaOH spray reagent for sugars. (b) R , = cm sample spot migrated/cm D-glucose migrated. glucose (d) Tailing which included darker areas at R . =0.42 and 0.32. glucose These darker areas were also reactive with a DMAB spray reagent (11)

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TABLE 6 Paper chromatography of acid-hydrolyzed TCA extracts/ IV. Sugars (internal standards) Standard r b glucose Glucose 1.00 Samples GSTA + glucose 0.94 0.42 d LSTA + glucose 0.98 0.43 d (a) Elution was ascending with solvent system B and spots were detected by the silver nitrate-NaOH spray reagent for sugars. ( b ) Glucose = Cm sam P le s P ot migrated/cm D-glucose migrated (c) To 50 ug of each sample spot, 5 ug of D-glucose was added as an internal standard. (d) Tailing which included darker areas similar to those in Table 5.

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35 Amino Acid and Amino Sugar Analysis of Crude Teichoic Acid When crude teichoic acid samples were subjected to amino acid and amino sugar analysis, the only major detectable peaks corresponded to glucosamine and galactosamine. In both types of teichoic acid samples (GS and LS) , the galactosamine was a minor but not negligible amount of the total hexosamine (GS , 18%; LS, 16%). There were traces of alanine in both samples, but there was too little to be quantified by the present method. These data raised the question of whether the carbohydrate found in acid hydrolysates was covalently attached to the presumed teichoic acid or not. Since the carbohydrate material consisted of neutral or cationic sugars, it was decided that the teichoic acid could be purified by DEAE chromatography. By allowing the anionic teichoic acid to stick to the DEAE groups, the carbohydrate, if not covalently linked, could be washed through the column. Then the teichoic acid could be recovered by eluting with a NaCl gradient. Crude Teichoic Acid Purification on DEAE-Sephadex Crude teichoic acids (GS, 142 mg; LS, 100 mg) were loaded on DEAE-Sephadex and washed with 0.1 M NaCl. A gradient of NaCl was used for elution (for specific details see Materials and Methods) . The washings and eluate were examined for the presence of phosphorus, reducing groups, and N-acetylhexosamine. The washings showed negligible amounts of either acid-molybdate or borate-DMAB reactive

PAGE 44

36 material. There was, however, some phenol-l^SO^ reactive material present. This amounted to about 5 ymoles of reducing groups for both GS and LS. The eluate profiles are shown in Figure 3. No reducing groups were found in these fractions. The major portion of the phosphate-hexosamine peaks eluted at NaCl concentrations of 0.73 M 0.82 M for the GS preparations and 0.85 M 0.91 M for the LS preparation. Both preparations had a smaller amount of material which eluted at much lower NaCl concentrations. Fractions 66-80 for LSTA and 43-60 for GSTA were pooled, dialyzed, and lyophilized. Dry weight recoveries were: GS-45.1 mg (32% of total material loaded on column) and LS-6.6 mg (7% of total material). Total phosphate recoveries were GS-70% (109 ymoles recovered from 156 ymoles applied to column) and LS-77% (109 ymoles recovered from 140 ymoles applied) . These purified teichoic acids were analyzed for total amino sugars, This analysis showed that there were 28% as many amino sugar residues as phosphate residues for the GS teichoic acid and 21% as many for the LS teichoic acid. By using a mild acid hydrolysis, it was possible to release the amino sugars with some of the N-acetyl groups originally present still intact. This approach yielded 47% (GS) and 54% (LS) of the total amino sugar as N-acetyl hexosamine. To further characterize the teichoic acids, they were analyzed by gel filtration.

PAGE 45

Figure 3. DEAE-Sephadex Chromatography of Crude Teichoic Acid Crude teichoic acids were applied to 9 g of DEAE Sephadex. The column was washed with 500 ml of 0.1 M NaCl. A salt gradient from 0.1 M to 1.0 M NaCl was used for elution. Fractions were assayed for total phosphate and N-acetylhexosamine.

PAGE 46

38 5

PAGE 47

jy Purified Teichoic Acid Characterization on Sephadex G-100 Purified teichoic acids (GS-42 mg, LS-6 mg) were run on Sephadex G-100 Fine in deionized water. The results of these runs are shown in Figure 4. K values for peak fractions were: 0.20 for LSTA and 0.71 and 0.69 for two separate isolations of GSTA (0.69 profile not shown) . These results suggested that the molecular weights of the teichoic acids might be relatively high and that the LS teichoic acid was substantially larger than the GS teichoic acid. To test this hypothesis, the average chain length was estimated. Chain Length Determination Purified teichoic acid was digested with bacterial alkaline phosphatase at pH 9.5 for 18 hours at 37°C to release terminal phosphate groups. The ratios of total phosphorus to labile phosphorus from two determinations are given in Table 7. Labile phosphorus was also measured for untreated purified teichoic acid and was not detectable. These results confirmed the prediction made by gel filtration that LS teichoic acid at about 70 glycerol phosphate units is, on the average, larger than GS teichoic acid at 38 units. With these basic characterizations accomplished, it was necessary to confirm the polyglycerol phosphate backbone structure by showing that a one-to-one ratio of glycerol and phosphate existed.

PAGE 48

Figure 4. Sephadex G-100 proctography of Purified Teichoic Acid. Puri fied teichoic acids were filtered « 160 ^of Sephadex G-100. Deionxzed water was used Total phosphate was measured for each ^a d plotted versus K values computed tor the P fractions.

PAGE 49

41

PAGE 50

TABLE 7 Molar ratios of labile and total phosphate in alkaline phosphatase digested, purified teichoic acids. 3 Sample Experiment number Total: labile phosphate Purified GSTA 1 38 2 38 Purified LSTA 1 66 2 74 (a) Teichoic acids were digested with bacterial alkaline phosphatase. After digestion inorganic and total phosphate were determined. (b) Values were determined by: Molar concentration of total phosphate/molar concentration of inorganic phosphate in digestion mixture.

PAGE 51

Molar Ratios of Glycerol and Phosphate in Purified Teichoic Acids Purified teichoic acids were acid hydrolyzed to break down the glycerol phosphate backbone into glycerol monoand di-phosphates, free glycerol, inorganic phosphate, and free amino sugars. The mixture was then treated with alkaline phosphatase. This mixture was assayed for free glycerol, inorganic and total phosphate. The molar ratios of glycerol to inorganic phosphate for two determinations are shown in Table 8. Analysis of total phosphate in these digested samples showed that alkaline phosphatase released 99% for GS and 87% for LS of the total bound phosphate. With this preliminary confirmation of the poly (glycerol phosphate) nature of the backbone it remained to be proven, that the amino sugars present were, in fact, covalently attached to the polyglycerol phosphate. This was accomplished by alkaline hydrolysis which breaks phosphodiester linkages but not glycosidic linkages. The general procedure used was similar to that of Van de Rijn and Bleiweis (83). Characterizations of Alkaline Hydrolysates Purified teichoic acid (GSTA only) was base hydrolyzed under conditions such as to break phosphodiester linkages without destroying glycosidic bonds. After neutralization, this hydrolysate was loaded onto a DEAE column to separate anionic compounds from cationic and neutral compounds. After the latter compounds were washed from

PAGE 52


PAGE 53

45 the column, the anionic compounds were eluted using a linear salt gradient. The profile of this elution is shown in Figure 5. The total phosphate represented in this profile was 58% (19 umoles) of the total phosphate (32.6 umoles) that was loaded on the column. The total amino sugar recovery from the elution was 87% (7.9 umoles) of the total amino sugar (9.1 umoles) that was loaded on the column. The ratio of total amino sugar to phosphate in the single peak of the total amino sugar was about 3.2 for ten fractions with the most amino sugar. The wash fraction contained the other 42% of the phosphate and 12% of the amino sugar. This fraction was further characterized by running on a Sephadex G-50 column. This profile is shown in Figure 6. The phosphate containing fractions (#36 #61) were pooled and lyophilized. More than 99% of the phosphate and the amino sugar was recovered from the gel filtration column. The lyophilized sample was diluted to 0.1 M NaCl and applied to a CM-Sephadex column in order to separate cationic molecules from neutral ones. After washing, the sample was eluted with a salt gradient and the profile is shown in Figure 7. Phosphate was also measured but none was detected (<10 nmoles/ml) . Amino sugar was, however, 100% recovered. The two amino sugar peaks, the pooled wash, and a fraction midway between the two amino sugar peaks were analyzed for glycerol. The pooled x^ash and the midway fraction contained no detectable glycerol. The smaller of the amino sugar peaks (fractions #4 and #5) contained one glycerol for every 2.9 amino sugar residues while the larger peak (fractions #41 and #42) contained one glycerol for every 6.5 amino sugar residues.

PAGE 54

Figure 5. Alkali-Hydrolyzed Teichoic Acid Elution on DEAE-Sephadex, Alkali-hydrolyzed GSTA was applied to 9 g of DEAE-Sephadex, Nonadherent compounds were removed with a 500 ml wash of 0.1 M NaCl. Adherent compounds were eluted with a linear 0,1 M to 1,0 M NaCl gradient. Fractions were assayed for total phosphate and total amino sugars.

PAGE 55

47 20 40 60 fraction no. 80

PAGE 56

Figure 6. Sephadex G-50 Chromatography of Neutral and Cationic Components of Alkali-hydrolyzed GSTA. The wash fraction from DEAE chromatography of alkalihydrolyzed GSTA was run on 124 ml of Sephadex G-50. Deionized water was used for elution. Fractions were assayed for total (P t ) and inorganic (Pj) phosphate, and total amino sugars.

PAGE 57

49 20 40 fraction no.

PAGE 58

3 "H W
PAGE 59

51 CO CN — |LU/ saioiuu^oi'JDBns ouiiuo 10404

PAGE 60

DISCUSSION Peptidoglycan Composition Amino acid and amino sugar analyses of purified wall hydrolysates first suggested the presence of wall associated polymers other than peptidoglycan in the wall of Artkrobacter arystallopoieies . The boiling SDS extraction used to purify walls is a very rigorous method which should remove all noncovalently associated cellular materials. Despite this, small amounts of the amino acids aspartic acid, threonine, glycine, and valine were detected (Table 2). These amino acids are not usually found in the pentapeptide of peptidoglycan. Also, there was more glucosamine than could be accounted for by peptidoglycan structure and there was galactosamine present which has not been reported to occur as part of the peptidoglycan. Krulwich et al . (55) have also reported on the amino acid and amino sugar composition of walls purified from A. avystallovoietes 15481. They also found small amounts of aspartic acid (0.15 mole/mole relative to glutamic acid in spheres, none found in rods) and glycine (0.14 mole/mole in spheres, 0.04-0.05 mole/mole in rods). They did not find valine or threonine but did find serine (0.13 mole/mole in spheres, none in rods) . It is conceivable that serine and threonine might be confused in some amino acid analyser programs since these amino acids are similar in structure. On the program used for my experiments, serine and threonine did elute next to each other but they were 52

PAGE 61

53 distinctly different peaks on standard runs. Krulwich et al. (56) used Ckzlaropsis B enzyme (N,0-diacetylmuramidase) (41), lysostaphin (a mixture containing endo-N-acetylglucosaminidase and a peptidase which releases N-terminal alanine and glycine from cell walls of Staphylococcus aureus strain Copenhagen, hydrolyzes polyglycine crossbridges, and, to a lesser extent, N-acetylmuramyl-L-alanine linkages) (15) , and AL-1 (a peptidase which breaks peptide crossbridges and N-acetylmuramyl-L-alanine linkages) (79) in separate experiments to obtain soluble fragemnts which were separated by ECTEOLA cellulose and gel filtration. N-terminal and Cterminal amino acids were then determined for various fragments. By these analyses, they came to the conclusion that aspartic acid and serine were not connected with either peptidoglycan or teichoic acid. They suggested that these amino acids were either contaminants or part of a small additional peptide in the wall. According to their results, the glycine present was a part of some of the crossbridges in spherical populations. The glycine present in rod populations was not, however, part of the peptidoglycan and presumably could also be a contaminant or part of an additional peptide. Tipper et al. (79) working in the same lab as Krulwich et al. also have reported on the cell wall composition of spherical .4. crystallopoietes . The composition they obtained is very similar to that of Krulwich et al. except that Tipper et al. reported about 0.03 umole/mg of wall of aspartic acid, serine, threonine, leucine, histidine, and methionine. Since their walls contained 0.42 umoles of total glutamic acid/mg of wall, 0.03 umole/mg of wall would be 0.07 moles/mole of glutamic acid. The nature of these trace

PAGE 62

54 amino acids and the ones which I have found is unclear but their presence calls for more research dealing with this problem. Krulwich et al. (55) found additional glucosamine (spheres, 2.09 mole/mole of glutamic acid; rods, 1.74-2.01 mole/mole) ,galactosamine (spheres, 0.31 mole/mole; rods, 0. 25-0. 30 mole/mole) .unidentified phenol-sulfuric acid-reactive material (spheres, 1.95 mole/mole; rods 1.04-1.37 mole/mole) , and phosphate (spheres, 2.02 mole/mole; rods, 1.88-2.12 mole/mole). Tipper et al. (41) also reported similar results for glucosamine and galactosamine. The walls analyzed in our laboratory also showed extra glucosamine (Table 1) , although the amount was generally less than that found by Krulwich et al. (55) or Tipper et al. (70). Quantitative differences in accessory polymers between the present data and past data are to be expected since, (1) the media used differ and, (2) the strains of bacteria used could be different because of extensive subculturing. Galactosamine was also found by our lab but it was not quantitated in this experiment since at the time the identity of that peak was unknown. Phenol-sulfuric acid-reactive material was also found in TCA extractable material but it was found that this material could be separated from teichoic acid by chromatography on DEAE-Sephadex. It seems likely that this material may contain the glucose which showed up in paper chromatography of acid hydrolysates of TCA extractable material. Krulwich et al. (55) were able to extract all the phosphate present in walls with 100% TCA at 60°C in less than one hour for both sphere and rod wall preparations. They concluded that this phosphate must be contained in teichoic acid since teichoic acids are extractable under those conditions. There are, however, other

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55 accessory polymers which have been discovered since the paper of Krulwich et al. (55) and which are extractable under conditions milder than those of Krulwich et al. (5,8,51,65). Tipper et al. (79) also found a phosphate polymer which co-eluted with some of the polysaccharide solubilized by Myxobaater AL-1 enzyme. Nevertheless, Krulwich et al. have turned out to be correct in their assumption even though today their data would not be considered sufficient to establish the presence of a teichoic acid as opposed to a sugar 1-phosphate polymer or a phosphorylated polysaccharide. Krulwich probably realized this later because in a paper a year after the one described above, Krulwich and Ensign (53) refer to the same moiety only as a "phosphate-containing polymer of the cell walls". Krulwich et al. (55) were correct about the lack of amino acid substitution of the teichoic acid. They found amino acids in their TCA extract but they were in the same ratios as were found in the peptidoglycan. They also incubated their purified cell wall in two alkaline buffers (pH 9.2 and pH 10.5) and found that no amino acids were released. This indicates that no amino acids are ester-linked to either polyols or to polysaccharides. In this work amino acid and amino sugar analyses of crude teichoic acids showed almost no amino acids present at all. The trace of alanine present could account for no more than one residue for every 100 glycerol phosphate residues in either GS or LS teichoic acid. It is more likely that this alanine arose from peptidoglycan contamination. Experimental data (see Table 2) suggest that all four non-pentapeptide amino acids can be removed if the right protease is used. This confirms the hypothesis that these amino acids are not part of

PAGE 64

56 the pentapeptide. This leaves the possibilities that these amino acids are contaminants, part of normal crossbridges, or part of a peptide attached to peptidoglycan. Trypsin is known to be a specific endopeptidase which breaks peptide bonds in which the carbonyl group is donated by lysine or arginine. Most peptidoglycan basic structures are notoriously resistant to trypsin (74), even those which contain penultimate L-lysine in the tetrapeptide (64). The reason for this resistance is not clear but certainly those L-lysine residues which are involved in crossbridges would not be susceptible due to a lack of positive charge on the E-amino group which is a necessary condition for trypsin digestion (49) . My data indicate that trypsin has very little if any effect on the level of alanine in purified cell walls. The alanine ratios remain fairly constant regardless of which proteolytic enzyme is used. The alanine ratios reported here agree fairly well with those of Krulwich et al . (55). The alanine in excess of the two moles in the tetrapeptide probably is due to the L-alanine crossbridges proposed by Krulwich et al . (55). By C-terminal analysis, they estimated that from 60 to 70% of the peptides are crosslinked, which also agrees with the amount of excess alanine reported here. If the effectiveness of the different proteolytic enzymes is compared, trypsin seems to be the most effective in removing the trace amino acids. The fact that trypsin removed most of these amino acids while a-chymotrypsin and pronase did not, may indicate that cleavage of a specific linkage to peptidoglycan is necessary to remove a small peptide. Also, it had earlier been noticed that if the concentration of pronase-digested wall to be analyzed was

PAGE 65

57 increased tenfold, a broad spectrum of non-pep tidoglycan amino acids appeared. This would seem to indicate that the trace amino acid represent only the most abundant amino acids present and, therefore, are the only ones normally detected since the levels of these nonpeptidoglycan amino acids were so low. Whether or not trypsin can remove or lower the amounts of these other amino acids is unknown. The possibility that the trace amino acids are no more than contaminants from membrane protein can not be discounted. This problem warrants further research. It is possible that by using a N-acetylmuramidse to solubilize the peptidoglycan, these amino acids could be characterized as belonging either to a high or low molecular weight fraction on gel filtration. Perhaps the most important fact in this experiment is that extra glucosamine and galactosamine were present. Combined with the presence of significant amounts of phosphate, this suggested the possibility that a teichoic acid was present. Since Krulwich et al. (55) had found that phosphate could be TCA extracted, a similar kinetic experiment was carried out but using the milder temperature (4°C as compared to 60°C that Krulwich et al. (55) used) normally used to extract teichoic acid with the least degradation (8,51). TCA Extraction As was shown in the Results section, the initial rate of extraction of phosphate was relatively high and was almost the same for GS and LS. By only 8 hours, about 73% of the total extractable phospahte had already been solubilized. These initially high rates of extraction

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5ET then dropped dramatically so that another 67 hours were required to remove the remaining 27% of the extractable phosphate. Thus, the average rate of extraction for the first 8 hours was about 9% of the extractable phosphate per hour whereas the average rate after 8 hours was about 0.4% per hour. This amounted to more than a 22-fold difference. The explanation for this difference in rates is not known but it does seem to indicate that there may be two different types of reactions occurring. After 75 hours of extraction, 81% of the GS and 53% of the LS total phosphate was solubilized. If the slow rate of extraction remained constant, it would take 5 days for GS and 8 days for LS phosphate to be completely extracted. Ghuysen et al. (34) reported that it took 3 weeks to extract more than 95% of the teichoic acid of Staphylococcus aureus. In the light of that study, it is likely that the rate of extraction does not remain constant but decreases further still with time. The actual dry weight of teichoic acid recovered after 96 hours of extraction varied depending on the method used for recovery of teichoic acid from TCA supernatants. If the weights recovered are corrected for the percentage recovery, then the actual weight of teichoic acid in the native wall (GS or LS) may be as high as about 43% (crude TA dry weight percentage of purified wall dry weight) . A small proportion of this crude teichoic acid is contaminating polysaccharide, so the actual figure is probably closer to around 40%. The amounts found in certain other gram positive bacteria range from 20% to 50% of the dry weight of the wall (8) .

PAGE 67

59 Characterization of Acid Hydrolysates To determine whether or not this phosphate-containing polymer was a teichoic acid, acid hydrolyses were carried out under conditions sufficiently rigorous to degrade a glycerol or ribitol teichoic acid to its monomers (3) . Table 3 shows the phosphate-containing monomers of acid-hydrolyzed crude teichoic acid. The ascending technique seems to give the least ambiguous results. The hydrolyzed teichoic acid spots matched fairly well with the mobility of 1,2 diphosphoglycerol. The fact that there were two spots in both the standards and the hydrolysates is probably due to some inorganic phosphate which should migrate a little farther than 1,2 diphosphoglycerol (50) and exists as a minor contamination in the 1,2 diphosphoglycerol standard. Hydrolyzed cardiolipin also showed a pattern of light and dark spots because this compound upon hydrolysis should give rise to 1,3 diphosphoglycerol (Rf, 0.12) and glycerol monophosphates (Rj, 0.35). These mobilities agree well with those found in the literature (7,29,50). The hydrolyzed teichoic acid spots on the descending run make it more difficult to choose between the 1,2 and the 1,3 compound. Indeed, the hydrolyzed teichoic acid samples may be a mixture of both which runs together on paper chromatography. This is actually what one would expect since a certain amount of ester migration can occur under acidic conditions (3) . It is noteworthy that no glycerol monophosphates were detected but the reason for this is not clear. In Table 4, it seems quite clear that the mobility of the hydrolyzed teichoic acid spots matched that of glycerol. The true picture,

PAGE 68

6tr however, is somewhat more cloudy than that. Anhydroribitol is reported to have the same mobility as glycerol under the conditions of chromatography used in this experiment. Apparently, the conditions of hydrolysis used for both the samples and the standard (2 N HC1, 100°C, 3 hours) were not sufficient to convert a detectable amount of ribitol to anhydroribitol. Anhydroribitol is reported to give a slow reaction with periodate-Schif f reagent (7) and so the fast reaction given by the hydrolyzed teichoic acid samples confirms that glycerol, not anhydroribitol, was present. The yellow spots which did not migrate were probably sugars which are characterized in Table 4. Both GS and LS crude teichoic acids appear to have the same sugars present as evidenced by the banding patterns obtained in the ethyl acetate-pyridine-water solvent system. Both preparations had a silver nitrate reactive spot which migrated the same distance as glucose. Both preparations also had darker areas in the long tails which extended from the origin to about halfway to the solvent front. These darker areas corresponded well to glucosamine and galactosamine standards. Later analyses confirmed the covalent linkage of these latter sugars to teichoic acid. The glucose is not connected with teichoic acid but may be part of the wall nevertheless. Since the spots that were presumably glucose did not exactly correspond in mobility to glucose in every run, glucose was added as an internal standard to the GS and LS acid-hydrolyzed preparations (Table 6) . In this chromatograph standard glucose and sample glucose

PAGE 69

61 migrated as a single spot. These spots moved slower than the glucose standard alone. The probable cause for this is the presence of small amounts of salt in the sample. Amino Acid and Amino Sugar Analyses of Teichoic Acid Amino acid and amino sugar analyses confirmed the identity of the glucosamine and galac to s amine indicated by paper chromatography. These analyses also indicated that there was about five times as much glucosamine present as galactosamine. The traces of alanine present would contribute less than one residue for every 100 glycerol phosphate residues. For this reason, it is felt that this alanine probably arose from contamination by peptidoglycan. DEAE Chromatography To purify the crude teichoic acids, ion exchange chromatography was used. The teichoic acid preparations were applied to the DEAESephadex under conditions which allowed the anionic teichoic acids to stick to the resin. Water washes removed a similar amount of phenol-suifuric acid reactive material from both preparations. This material is presumed to contain the glucose present in crude teichoic acid samples as demonstrated by paper chromatography. Lyophilization of this material rendered it water-insoluble and so no further analyses of this material were carried out. No phenol-H2S0 4 reactive material

PAGE 70

^T was found in the salt-eluted fractions, so glucose is presumed not to be part of the teichoic acid structure. The profiles of Figure 3 show that GSTA and LSTA are different in their affinity for DEAE groups. The peaks of phosphate eluted at salt concentrations which were about 0.1 M different. This suggests that there may be a difference in net charge of the molecules being separated. These profiles also show that the amino sugar curve closely matched that of phosphate. If the ratios between phosphate and amino sugar are calculated for each fraction in the main peaks, these ratios are relatively constant. They are so constant that in more than 95% of the cases, the differences can be attributed to a change from the mean ratio of an amount that can be accounted for by a change of one or two residues. Thus suggests that these two moieties were associated with each other and not simply eluting close to one another. Dry weight recoveries were quite low as compared to phosphate recoveries. This is probably due more to inaccuracies in dry weight measurement (resulting from incomplete water removal from samples) than to a removal of contamination. Acid Hydrolyses of Purified Teichoic Acids The amount of amino sugar in the two preparations was different but not radically so. The mild acid hydrolysis only demonstrated that about one half of the amino sugars were N-acetylated, but this is strictly a minimum figure. Even under these mild conditions some hydrolysis of the N-acetyl groups probably occurs. It is therefore

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probable that most amino amino sugar was N-acetylated. DMAB reagent showed a limited reactivity to amino sugar associated with teichoic acid because the N-acetyl amino sugar is bound at the C-l position. N-Acetylation with acetic anhydride did not increase this reactivity. This would seem to indicate that all the amino sugars are already N-acetylated. When the teichoic acid was acid hydrolysed a large increase in reactivity to DMAB occurred due to exposure of C-l. Gel Filtration Teichoic acids were also characterized by gel filtration. It was found necessary to use Sephadex G-100, a gel with a molecular weight exclusion limit much higher than any known teichoic acid molecular weight. This is probably due to the high charge and extended shape common to teichoic acids (25,27). The profiles in Figure 4 again show that there was a considerable difference between GSTA and LSTA. The most obvious cause for this is a difference in molecular weight but if there were a large difference in charge or shape of the molecule, it could affect the retention of the teichoic acid molecules. Determination of Chain Length To further characterize the teichoic acids and to determine if molecular weight was the determining factor in the gel filtration profile, chain lengths were determined. Average chain length can be determined by releasing the terminal phosphomonoester group

PAGE 72

64 with alkaline phosphatase and measuring the inorganic phosphate released and comparing that to the total phosphate content. This gives the average number of glycerol phosphate residues per chain if there is a one-to-one correspondence of glycerol to phosphate. These determinations showed that GSTA at 38 glycerol phosphate units was only about half as long as LSTA at an average of 70 units. These numbers are representative of purified teichoic acid preparations only and may not reflect accurately the native chain lengths as found in the wall (8) . TCA is known to break phosphodiester bonds (77) . Phosphodiester bonds not only link the glycerol phosphate monomers, they are also responsible for linking the teichoic acid to peptidoglycan in the only well-characterized cases to date (22,38,77). It is not surprising then that a certain amount of degradation of chain length would occur upon TCA extraction of teichoic acids. Indeed, this has been observed in several cases (8,34,51). The kinetic data seem to indicate that there may be two mechanisms of extraction. This may be reflective of the phosphodiester links which bond the backbone units and those which may link the backbone to peptidoglycan. Even so, the difference between GSTA and LSTA would be hard to explain by differing labilities to acid extraction when their composition is so similar. Thus, even if the chain lengths do not accurately reflect those in the native wall, there still should be a significant difference between the lengths of GSTA and LSTA in the wall. It is very probable that the differing chain lengths of GSTA and LSTA account for some of the differences observed in ion-exchange chromatography. DEAE chromatography has been shown to separate oligonucleotides according to net negative charge which is a function

PAGE 73

65 of chain length under conditions minimizing charge and secondary binding forces of the purine and pyrimidine bases (72,81,85). It is reasonable to assume then that LSTA with an average of 70 negative charges per molecule is going to be eluted at a higher ionic strength than is GSTA which only has as average of 38 negative charges per molecule. This is, in fact, what is observed (Figure 3). This reasoning can be extended to the material which elutes at lower ionic strengths which would be of lower chain length than the bulk of the material. This material probably resulted from degradation by TCA hydrolysis of backbone phosphodiester bonds. The difference in chain length also is reflected in the gel filtration profile but it should be noted that the difference in charge per molecule may also play a role in the behavior of teichoic acids on Sephadex G-100. Pharmacia states that Sephadex G-100 contains small numbers of carboxyl groups and, therefore, at low ionic strengths, negatively charged moleclues may be excluded from the gel to a greater extent than an uncharged molecule of the same molecular weight (67). Glycerol-Phosphate Ratios Many of the above conclusions can only be drawn by assuming that there is a poly (glycerol phosphate) backbone. To confirm the presence of such a backbone, it is necessary to show a one-to-one ratio between glycerol and phosphate. As Table 8 shows, this is the case for both GSTA and LSTA.

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66 Alkaline Hydrolysis The final characterization of teichoic acid was by alkaline hydrolysis. The primary goal of this experiment was to establish whether or not the amino sugars were covalently attached to teichoic acid. Alkaline hydrolysis leaves glycosidic linkages intact while cleaving phosphodiester linkages where free hydroxyl groups are available on neighboring carbons allowing for a cyclic phosphate intermediate to form (50). Figure 5 shows that a substantial amount of amino sugar elutes from DEAE-Sephadex as a single peak. Since only anionic molecules should be bound to this resin, this suggests tha the amino sugar is still bound to glycerol phosphate. This peak accounts for 87% of the amino sugar in the alkali-hydrolyzed GSTA. Thus, the majority of amino sugar is covalently attached to teichoic acid and it suggests that the poly (glycerol phosphate) backbone is 2,3 linked rather than 1,3 linked. In a 1,3 teichoic acid if a glycosidic bond ties up C-2 of a glycerol phosphate residue, then there is no free hydroxyl on that residue for the necessary cyclic phosphate to form. For this reason, one expects only glycosylglycerol for substituted residues. In contrast, glycosylglyceromonophophates can only result from a 2,3linked teichoic acid structure. It has never been found in membrane teichoic acids and only in a few cases in wall teichoic acids (32,63). The amount of amino sugar exceeds the amount of phosphate in the amino sugar peak by a factor of about 3.2. This suggests that there may be an average of three amino sugars linked to each other by

PAGE 75

67 glycosidic bonds and this trimer is attached to a single glycerol phosphate residue. The other 12% of the amino sugar in the alkaline-hydrolyzed GSTA was characterized as being low molecular weight because it was almost completely included in a Sephadex G-50 gel (Figure 6) . This material was then eluted on a cation exchange resin. All the remaining phosphate was washed through the column with 0.1 M NaCl. Two peaks of amino sugar were then eluted using a linear NaCl gradient. Together these accounted for all of the amino sugar from the DEAE wash fraction. No phosphate was detected in either fraction. 3oth peaks did contain glycerol. The smaller peak contained about three amino sugar residues for each glycerol residue while the larger peak contained 6.5 amino sugars per glycerol. It would seem, therefore, that CM-Sephadex acts in manner analogous to DEAE-Sephadex in that it can separate positively charged molecules with similar charge densities by chain length. These data also suggest that amino sugar is covalently attached to teichoic acid and may exist as trimers or hexamers attached to single glycerol residues. The significance of the larger amount of the hexamer in this relatively small fraction of amino sugar is not known. Although these results were obtained only for GSTA, it seems reasonable that LSTA follows a similar but not identical pattern.

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68 Summary of Teichoic Acid Structure Arthrobacver arystallopoietes contains a glycerol wall teichoic acid in both sphere and rod morphologies in approximately equal amounts. This teichoic acid is 38 glycerol phosphate units long in spheres and about 70 units long in rods. N-Acetylhexosamine (N-acetylglucosamine, about 83% and N-acetylgalactosamine, about 17%) is attached by a glycosidic linkage from CI of the hexosamine to CI of glycerol. On the average, there are 11 hexosamines per chain in sphere teichoic acid while there are about 15 hexosamines per chain in the rod teichoic acid. These hexosamines may exist in polymerized side chains which have three sugars on the average. Thus, only about 11% (GS) or 7% (LS) of the glycerol phosphate units are substituted. The poly (glycerol phosphate) backbone is 2,3 linked for GSTA and probably for LSTA also. This is the first teichoic acid that has been characterized to this extent in the genus Arthrobaatev . Possible Roles for Teichoic Acids in Avthrobaater One role of teichoic acids that is well established is binding of cations, particularly divalent cations ( 6,10, 26,42 , 46) . Evidence has been presented that wall and membrane teichoic acids provide a controlled reservoir of bound Mg + 2 for membrane bound enzymes (46) which require it for maximal activity (2,21,66). It has been suggested that the wall teichoic acid scavenges Mg +i from the environment and transfers it to lipoteichoic acid which interacts with the membrane

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69 enzymes. Baddiley et al. (10) have shown that alanylated teichoic acid interacts less strongly with Mg +2 and, therefore, the degree of D-alanine substitution may act as a controlling influence on Mg +2 binding. It seems reasonable that the teichoic acid in A. aystallopo-ietes could serve the same function since its structure is similar to some of those used in the above mentioned studies. If this were true, then it could be that the longer chains found in rod shaped bacteria might more effectively scavenge Mg +2 than the short chains of the spheres. This would be consistent with the higher growth rate of rods which would presumably necessitate a greater flux of Mg to the membrane. Another function of teichoic acids is suggested by the studies of Burge et al. (20) and Milward and Reavely (62). By examination of peptidoglycans containing teichoic acid and those which had teichoic acid artificially removed, these authors found that extraction of teichoic acid affects the flexibility of the peptidoglycan. It was suggested (20) that since teichoic acid can pass through the pores in the outer layers of peptidoglycan it would resist shear between adjacent peptidoglycan sheets and still allow for expansion or contraction of the wall. The most interesting function as far as this work is concerned is suggested by several studies in which teichoic acid changes effect autolytic enzymes. Holtje and Tomasz (45,80) have described an N-acetylmuramyl-L-alanine amidase of Pneicmocooaus which required choline residues in the wall teichoic acid for maximal binding. If ethanolamine residues were

PAGE 78

70 substituted, the cell walls were resistant to the autolysin. Besides lowered binding of the autolysin to the wall, this effect was due to the inability of the altered teichoic acid to convert the autolysin to its active form. Herbold and Glaser (43) have described a similar Nacetylmuramyl-L-alanine amidase in Bacillus subtilis . Again cell walls lacking teichoic acid had a much lower affinity for the enzyme than those with normal levels of teichoic acid. In a different strain of Bacillus subtilis, Brown et al. (16,18) had such trouble purifying autolysin from teichoic acid that they suggested that the two molecules might be covalently attached to each other. They speculated that the close association between autolysin and the teichoic acid might serve to localize the autolysin for maximum utilization of substrate. In a temperature conditional morphological mutant of Bacillus subtilis, Boylan et al. (12,13,24) found that at the nonpermissive temperature teichoic acid was deficient. These mutants grew as normal rods at 30°C but changed to irregular spheres at 45°C. The walls of the spheres were also deficient in N-acyl muramyl-L-alanine amidase. Rogers et al. have found similar results in seven rod A mutants of Bacillus subtilis (17,71) and Bacillus liche.nifovm.is (33). Other authors (44,75) have also suggested that autolysins may play a significant role in the determination of morphology in all bacteria. In A. crystallcpoietes it has been shown that the activity of N-acetylmur amidase correlates with morphogenesis (53) . The high activity of the autolysin in spheres results in the relatively short glycan backbones found in the peptidoglycan. In the same way, the low activity of this enzyme in rod walls results in relatively

PAGE 79

71 long glycans. The length of the glycan chains probably influences the rigidity of the peptidoglycan and, therefore, the morphology of the organism (55) . The present work has shown that there is almost a two-fold difference in the length of teichoic acids isolated from spherical and rod-shaped A. erystallopoietes. In light of the correlations between morphology, autolysins, and teichoic acids discussed above, it does not seem unreasonable that this difference may play a role in morphogenesis. Phosphate analyses of purified wall show that there is slightly more phosphate per mg of rod wall than in sphere wall (rods, 21 ug/ml wall; spheres, 18 ug/mg wall). Since the rod teichoic acid has almost twice the phosphate per chain as the sphere teichoic acid because of its length, this means that there are about one half as many rod teichoic acid chains as there are sphere teichoic acid chains. If autolysin were binding to the teichoic acid chains, the rod wall would present about one half as many binding sites proximal to the wall as would the sphere wall. A question that arises from this model is why the autolysin does not cleave the N-acetylmuramyl-N-acetylglucosaminyl residue in peptidoglycan to which it is attached. This would be disadvantageous to the cell because both teichoic acid and autolysin would be lost from the cell which represents a considerable energy investment wasted. One possible solution to this problem could lie in the specificity of the autolysin. The autolysin in A. avystallopo-ietes thought to be responsible for control of glycan length is an N-acetylmuramidase. It is possible that this enzyme can not hydrolyze at the reducing

PAGE 80

72 end of an N-acetylmuramic acid which has the C-6 hydroxyl tied up in covalent linkage. Lysozyme is known to be inhibited under these conditions and teichoic acids are known to inhibit lysozyme in certain cases (78). The only well characterized linkages of teichoic acid to peptidoglycan are to the C-6 hydroxyl of N-acetylmuramic acid. In both our laboratory and that of Krulwich et al. (55), lysozyme was found to be poorly lytic against purified cell walls of A. cvystallopoietes. Chalaropsis B enzyme, on the other hand, completely solubilizes these walls. Chalaropsis B enzyme is an N,0-diacetylmuramidase which is similar to lysozyme except that it will cleave at the reducing ends of N-acetylmuramic acids which are substituted in the C-6 position. So, apparently something is attached at this position in .4. cvystallovoietes. It is possible that this might be teichoic acid but it could also be polysaccharide. If it were teichoic acid, it may be the reason why autolysin does not remove teichoic acid. Krulwich and Ensign (53) did find that the autolysin preferentially solubilizes peptidoglycan which is low in phosphate content. Another reason for this result might be steric hindrance of the enzyme by the teichoic acid. It may be that teichoic acid does not bind the autolysin at all but only serves in substrate recognition by the enzyme as suggested by Higgins and Shockman (44) . In Streptococcus faecalis, the N-acetylmuramidase binds to TCA extracted walls but these walls are hydrolyzed slower by autolysin than untreated walls. The TCA extracted walls are, however, a better substrate for lysozyme.

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73 It is also possible that the divalent cation binding property of teichoic acids is important in regulating the autolysin. Clearly, there is much more to be found out about the roles of teichoic acids in .4. cvystallovoietes. Hopefully, further research will shed light on these roles.

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LITERATURE CITED 1. Ames, B.N. 1966, Assay of inorganic phosphate, total phosphate and phosphatases. Meth. in Enzymol. 8: 115-118. 2. Anderson, R.G., H. Hussey, and J, Baddiley. 1973. The mechanism of wall synthesis in bacteria. Biochem. J. 227:11-25. 3. Archibald, A.R. and J, Baddiley. 1966. The teichoic acid. Adv. Carbohydrate Chem. 21:323-375. 4. Archibald, A.R., J. Baddiley, and N.L, Blumson. 1968. The teichoic acids, Adv. Enzymol. 5/7:223-253. 5. Archibald, A.R., J. Baddiley, D. Button, S. Heptinstall, and G.H. Stafford. 1968. Occurrence of polymers containing N-acetylglucosamine 1-phosphate in bacterial walls. Nature (London) 22 9:855-856. 6. Archibald, A.R., J. Baddiley, and S. Heptinstall. 1973. The alanine ester content and magnesium binding capacity of walls of Staphylococcus aureus H grown at different pH values. Biochim. et Biophys." Acta 213:629-634. 7. Armstrong, J.J., J. Baddiley, J.G. Buchanan, B. Carss, and G.R. Greenberg. 1958. Isolation and structure of ribitol phosphate derivatives (teichoic acids) from bacterial cell walls. J. Chem. Soc. (London), p. 4344-4354. 8. Baddiley, J. 1972. Teichoic acids in cell walls and membranes of bacteria. Essays in Biochem. 3: 35-77, 9. 3addiley, J., J.G. Buchanan, R.E. Handschumacher , and J.F. Prescott. 1956. Chemical studies in the biosynthesis of purine nucleotides. Part I. The preparation of N-glycylglycosylamines. J. Chem. Soc. pp. 2818-2823. 10. Baddiley, J., I.e. Hancock, and P.M. A. Sherwood. 1973. X-ray photoelectron studies of magnesium ions bound to the cell walls of grampositive bacteria. Nature (London) 243: 43-45. 11. Belcher, R. , A.J. Nutten, and CM. Sambrook. 1956. The determination of glucosamine. Analyst 73:201-208. 12. Boylan, R.J. and N.H. Mendelson. 1969, Initial characterization of a temperature-sensitive rod" mutant of Badlluo subvilis. J. 3acteriol. 2 55:1316-1321., 74

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75 13. Boylan, R.J., N.H. Mendelson, D. Brooks, and F.E. Young. 1972. Regulation of ^the bacterial cell wall: Analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid. J. Bacteriol. 110:281-290. 14. Braun, V. and U. Sieglin. 1970. The covalent murein-lipoprotein structure of the Escherichia coli cell wall; the attachment site of the lipoprotein on the murein. Eur. J. Biochem. 13:336-346. 15. Browder, H.P., W.A. Zygmunt, J.R. Young, and P. A. Tavormina. 1965. Lysostaphin: Enzymatic mode of action. Biochem. Biophys. Res. Comm. 75:383-389. 16. Brown, W.C., D.K. Jraser, and F.E. Young. 1970. Problems in purification of a Bacillus subtilis autolytic enzyme caused by association with teichoic acid. Biochim. Biophys. Acta 135:308-315. 17. Brown, W.C., C.R. Wilson, S. Lukehart, F.E. Young, and M.A. Shiflett. 1976. Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis. J. Bacteriol. 125:166-173. 18. 19. Brown, W.C. and F.E. Young. 1970. Dynamic interactions between cell wall polymers, extracellular proteases and autolytic enzymes. Biochem. Biophys. Res. Comm. 35:564-568. Brundish, D.E., N. Shaw, and J. Baddiley. 1965. The occurrence of glycolipids in gram-positive bacteria. Biochem. J. 35 :21c-22c. 20. 3urge, R.E., A.G. Fowler, and D.A. Reaveley. 1977. Structure of the peptidoglycan of bacterial cell walls. I. J. Mol. Biol. Ii7:927-953. 21. Burger, M.M. and L. Glaser. 1964. The synthesis of teichoic acids. I. Polyglycerophosphate. J. Biol. Chem. 233:3168-3177. 22. Button, D., A.R. Archibald, and J. Baddiley. 1966. The linkage between teichoic acid and glycosaminopeptide in the walls of a strain of Staphylococcus lactis. Biochem. J. 99 :llc-14c. 23. Chen, P.S., Jr., T.Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. 25:1756-1758. 24. Cole, R.M., T.J. Popkin, R.J. Boylan, and N.H. Mendelson. 1970. Ultrastructure of a temperature-sensitive rod mutant of Bacillus subtilis. J. Bacteriol. 753:793-810. 25. Critchley, P., A.R. Archibald, and J. 3addiley. 1962. The intracellular teichoic acid from Lactobacillus arabinosus 17-5. Biochem. J. 55:420-430. 26. Cutinelli, C. and F. Galdiero. 1967. Ion-binding properties of the cell wall of Staphylococcus aureus. J. 3acteriol. 33:2022-2023.

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76 28 27. Doyle, R.J. , M.L. McDannel, U.N. Streips, D.C. Birdsell, and F.E. Young. 1974. Polyelectrolyte nature of bacterial teichoic acids. J. Bacterid. 125:606-615. Dubois, K.A., K.A. Gilles, J.K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 23:350-356. 29. Ellwood, D.C, M.V.Kelemen, and J. Baddiley. 1963. The glycerol teichoic acid from the walls of Staphylococcus albus N.T.C.C. 7944. Biochem. J. 3(3:213-225. 30. Ensign, J.C. and S.C. Rittenberg. 1963. A crystalline pigment produced from 2-hydroxypyridine by Arthrcbacter crystallopoietes n. sp. Arch. Mikrobiol. 47:137-153. 31. Ensign, J.C. and R.S. Wolfe. 1964. Nutritional control of morphogenesis in Arthrobactsr crystallopoietes . J. Bacterid. 37:924-932. 32. Forrester, I.T. and A.J. Wicken. 1966. The chemical composition of cell walls of some thermophilic bacilli. J. Gen. Microbiol. 42:147-154. 33. Forsberg, C.W. , P.B. Wyrick, J.B. Ward, and H.J. Rogers. 1973. Effect ^of phosphate limitation on the morphology and wall composition of Bacillus licheniformis and its phosphoglumutase-def icient mutants. J. Bacteriol. 213:969-984. 34. Ghuysen, J.M. , D.J. Tipper, and J.L. Strominger. 1965. Structure of the cell wall of Staphylococcus aureus, strain Copenhagen. IV. The teichoic acid-glycopeptide complex. Biochemistry 4:474-485. 35. Ghuysen, J.M. , D.J. Tipper, and J.L. Strominger. 1966. Enzymes that degrade bacterial cell walls. Meth. in Enzymol. 3:115-118. 36. Goldstein, I.J. and W.J. Whelan. 1962. Structural studies of dextrans. Part I. A dextran containing a-1 ,3-glcosidic linkages. J. Chem. Soc. (London), p. 170-175. 37. Goodwin, S.D. and J.G. Shedlarski. Jr. 1975. Purification of cell wall peptidoglycan of the dimorphic bacterium Caulobactev crescentus. Arch. Biochem. 3iophys. 170:23-36. 38. Grant, W.D. and A.J. Wicken. 1968. Muramic acid phosphate and the linkage of teichoic acid to peptidoglycan in Bacillus stearothermophilus cell walls. Biochem. Biophys. Res. Comm. -32:122-128. 39. Hamilton, R.W. , E.C. Achberger, and P.E. Kolenbrander . 1977. Control of morphogenesis in Arthrcbaater crystallopoietes: Effect of cyclic adenosine 3 ' ,5 '-monophosphate. J. Bacteriol. 225:874-879.

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77 40. Hanes, C.S. and F.A. Isherwood. 1949. Separation of the phosphoric esters on the filter paper chromatogram. Nature (London) J o^: 11071112. 43. 44, 41. Hash, J.H. and M.V. Rothlauf. 1967. The N,0-diacetylmuramidase of Chalaropsis species. I.: Purification and crystallization. J. Biol. Chem. 242:5586-5590. 42. Heptinstall, S. , A.R. Archibald, and J. Baddiley. 1970. Teichoic acids and membrane function in bacteria. Nature (London) 225:519521. Herbold, D.R. and L. Glaser. 1975. Bacillus subtilis N-acetylmuramic acid L-alanine amidase. J. Biol. Chem. 25(9:1676-1682. Higgins, M.L. and G.D. Shockman. 1971. Procaryotic cell division with respect to wall and membranes. In Critical Reviews in Microbiology. Chemical Rubber Co. p. 29-72. 45. Holtje, J.-V. and A. Tomasz. 1975. Specific recognition of choline residues in the cell wall teichoic acid by the N-acetylmuramyl-Lalanine amidase of Pneumococcal . J. Biol. Chem. 255:6072-6076. 46. Hughes, A.H., I.C. Hancock, and J. Baddiley. 1973. The function of teichoic acids in cation control in bacterial membranes. Biochem. J. 132:83-93. 47. Jacobsen, R.A. and L.F. Mazzuckelli. 1973. Evidence for two RNA polymerases in Arthrobacter , a morphogenetic bacterium. Biochem. Biophys. Res. Coram. 3:867-873. 48. Keddie, R.M. 1974. Genus II. Arthrobacter Conn and Dimmick 1947, 300. In Sergey's Manual of Determinative Bacteriology, 8th edn. p. 618-625. Edited by R.E. Buchanan and N.E. Gibbons. Baltimore: Williams & Wilkins. 49. Keil, B. 1971. Trypsin. In The enzymes, 3rd edn. p. 249-275. 50. Kelemen, M.V. and J. 3addiley. 1961. Structure of the intracellular glycerol teichoic acid from Lactobacillus casei A.T.C.C. 7469. Biochem. J. 30: 246-254. 51. Knox, K.W. and A.J. Wicken. 1973. Immunological properties of teichoic acids. Bacteriol. Rev. 37:215-257. 52. Kolenbrander, P.E. and M. Weinberger. 1977. 2-Hydroxypyridine metabolism and pigment formation in three Arthrobacter species. J. Bacteriol. 132:51-59. 53. Krulwich, T.A. and J.C. Ensign. 1968. Activity of an autolytic M-acetylmuramidase during sphere-rod morphogenesis in Arthrobacter crystallopoietes. J. Bacteriol. 96:857-859.

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78 54. Krulwich, T.A. and J.C. Ensign. 1969. Alteration of glucose metabolism of Arthrobaoter crystallopoietes by compounds which induce sphere to rod morphogenesis. J. Bacteriol. 37:526-534. 55. Krulwich, T.A., J.C. Ensign, D.J. Tipper, and J.L. Strominger. 1967. Sphere-rod morphogenesis in Arthrobaoter crystallopoietes. I. Cell wall composition and polysaccharides of the peptidoglycan. J. Bacteriol. 5^:734-740. 56. Krulwich, T.A., J.C. Ensign, D.J. Tipper, and J.L. Strominger. 1967. Sphere-rod morphogenesis in Arthrobaoter crystallopoietes. II. Peptides of cell wall peptidoglycan. J. Bacteriol. 34:741-750. 57. Kuhn, R. , M.P. Starr, D.A. Kugn, H. Bauer, and H.-J. Knackmuse. 1965. Indigoidine and other bacterial pigments related to 3,3'bipyridyl. Arch. Mikrobiol. 52:71-84. 58. Lowry, O.H. , N.R. Roberts, K.Y. Leiner, M.-L. Wu, and A.L. Farr. 1954. The quantitative histochemistry of brain. I Chemical methods. J. Biol. Chem. 207:1-17. 59. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 60. Luscombe, B.M. and T.R.G. Gray. 1971. Effect of varying growth rate on the morphology of Arthrobaoter. J. Gen. Micro. 53:433-434. 61. Massey, L.K. , J.B. Clark, and R.A. Jacobsen. 1973. Changes in RNA transcription during morphogenesis of Arthrobaoter crystallopoietes. J. Gen. Microbiol. 77:51-60. 62. Millward, G.R. and D.A. Reaveley. 1974. Electron microscope observations on the cell walls of some gram-positive bacteria. J. Ultrastruct. Res. 4o:309-326. 63. Naumova, 1.3. and M.Z. Xaretskaya. 1964. Some properties of glyceroteichoic acids from Streptomyces rimosus T-118 and Strevtomyoes antibiotious 39. Dokl. Akad. Nauk. SSSR. 257:207-210. 64. Park, J.T. and R. Hancock. 1960. A fractionation procedure for studies of the synthesis of cell-wall mucopeptide and of other polymers in cells of Staphylococcus aureus. J. Gen. Microbiol. 52:249-258. 65. Partridge, M.D., A.L. Davison, and J. Baddiley. 1971. A polymer of glucose and N-acetylglucosamine 1-phosphate in the wall of Micrococcus sp. Al. Biochem. J. 222:695-700. 66. Patterson, P.H. and W.J. Lennarz. 1971. Studies on the membranes of bacilli. I. Phospholipid biosynthesis. J. Biol. Chem. 246:10621072.

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79 67. Pharmacia booklet "Sephadex-gel filtration in theory and practice." 1970. p. 11. 68. Previc, E.P. 1970. Biochemical determination of bacterial morphology and the geometry of cell division. J. Theor. Biol. 27:471-479. 69. Previc, E.P. and N. Lowell. 1975. Peptidoglycan compositions of a new strain of Arthrobacter crystallovoietes during sphere-rod morphogenesis. Biochem. et Biophys. Acta 422:377-385. 70. Reissig, J.L., J.L. Strominger, and L.F. Leloir. 1955. A modified colorimetric method for the estimation of X-acetylamino sugars. J. Bio. Chem. 227:959-966. 71. Rogers, H.J. and C. Taylor. 1978. Autolysins and shape change in Rod A mutants of Bacillus subtilis. J. Bacteriol. 255:1032-1042. 72. Rushizky, G.W. , E.M. 3artos, and H.A. Sober. 1964. Chromatography of mixed oligonucleotides on DEAE-Sephadex. Biochemistry 5:626-629'. 73. St. John, A.C. and J.C. Ensign. 1976. Macromolecular synthesis and cell division during morphogenesis of Arthrobacter cry stallovoietes . Arch. Microbiol. 222:51-58. 74. Saiton, M.R.J. 1953. Cell structure and the enzymic lysis of bacteria. J. Gen. Microbiol. 5:512-523. 75. Schleifer, K.H., W.P. Hammes, and 0, Kandler. 1976, Effect of endogenous and exogenous factors on the primary structures of bacterial peptidoglycans. Adv. Microbial Physiol. 25:245-292. 76. Spackman, D.H., W.H. Stein, and S. Moore. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem. 55:1190-1206. 77. Strominger, J.L. and J, -M. Ghuysen. 1963. On the linkage between teichoic acid and the glycopeptide in the cell wall of Staphylococcus aureus. Biochem. Biophys. Res. Comm. 22:418-424. 73. Strominger, J.L. and J. -M. Ghuysen. 1967. Mechanisms of enzymatic bacteriolysis. Science 156: 213-221. 79. Tipper, D.J., J.L. Strominger, and J.C. Ensign. 1967. Structure of the cell wall of Staphylococcus aureus strain Copenhagen. VII. Mode of action of the bacteriolytic peptidase from Myxobacter and the isolation of intact cell wall polysaccharides. Biochemistry o:906-920. SO, Tomasz, A. 1968. Biological consequences of the replacement of choline by ethanolamine in the cell wall of Pneumococeus : Chain formation, loss of transf ormability, and loss of autolysins. Proc. Natl. Acad. Sci, U.S.A. 5.9:86-93.

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80 81. Tomlinson, R.V. and G.M. Tener. 1963. The effect of urea, formamide, and glycols on the secondary binding forces in the ion-exchange chromatography of polynucleotides on DEAE-Cellulose. Biochemistry 2:697-702. 12. Trevelyan, W.E., D.P. Procter, and J.S. Harrison. 1950. Detection of sugars on paper chromatograms. Nature (London) 186; 444-445. 83. Van De Rijn, I. and A.S. Bleiweis. 1973. Antigens of Streptococcus mutans. I. Characterization of a serotype-specif ic determinant from Streptococcus '-nutans. Infect. Immunity 7:795-804. 84. Ward, CM., Jr. and G.W. Claus. 1973. Gram characteristics and wall ultrastructure of Arthrobacter crystallopoietes during coccus-rod morphogenesis. J. Bacteriol. 214:378-389. 85. Yogo, Y. and E. Wimmer . 1973. Poly(A) and poly (U) in poliovirus double stranded RNA. Nature New Biol. 242:171-174.

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BIOGRAPHICAL SKETCH John H. Hellmuth was bom September 3, 1952, in Jacksonville, Florida. There he attended The Bolles School and graduated in May, 1970. In December, 1973, he received the degree of Bachelor of Science with honors, with a major in biological sciences with an emphasis in Microbiology from North Carolina State. From January, 1974, until the present time, he has pursued his work toward the degree of Doctor of Philosophy in the Department of Microbiology and Cell Science, University of Florida. John H. Hellmuth was married August 25, 1973, to the former Martha Jean Payne of Jacksonville, Florida, and is the father of a daughter, Kelly Leigh.

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. JAtjji r Edward P. Previc, Chairman Associate Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. // ' V /-'
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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Lonnie 0. Ingram $ " ~" Associate Professor of Microbiology and Cell Science This dissertation was submitted to the Graduate Faculty of the Department of Microbiology and Cell Science in the College of Liberal Arts and Sciences and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 1978 9ean, Graduate School

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UNIVERSITY OF FLORIDA 3 1262 08553 2751